Live-attenuated vaccine having mutations in viral polymerase for the treatment and prevention of canine influenza virus

ABSTRACT

The present invention relates to compositions and methods for the treatment and prevention of canine influenza virus (CIV) and CIV-related pathology. The present invention is based in part upon the discovery that various mutations in segment 1 and segment 2 of the CIV genome, thereby encoding mutant PB2 and PB1 protein, render the virus to be temperature-sensitive.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a U.S. national phase application filed under 35U.S.C. § 371 claiming benefit to International Patent Application No.PCT/US16/47715, filed Aug. 19, 2016, which is entitled to priority under35 U.S.C. § 119(e) to U.S. Provisional Patent Application No.62/207,571, filed Aug. 20, 2015, each of which application is herebyincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Influenza A viruses (IAVs) are enveloped viruses that belong to theOrthomyxoviridae family, and contain a genome comprised of eightsingle-stranded negative-sense RNA viral segments that encode for 10-14proteins (Baker et al., 2015, Future Virology, 10: 715-730). Thehemagglutinin (HA) and the neuraminidase (NA) glycoproteins are themajor antigenic determinants of IAV and are essential for receptorbinding and fusion, and virion release, respectively (Varghese et al.,1992, Proteins, 14:-327-332). IAV HA and NA glycoproteins withininfected organisms and populations are driven to evolve antigenicvariants via immunologic pressure, and positive selection of fit virusesoccurs gradually in a process known as antigenic drift (Carrat et al.,2007, Vaccine, 24: 6852-6862). The antigenic diversity of glycoproteinsis used to further classify IAVs, of which there are 18 HA and 11 NAsubtypes (Palese, 2007, The Viruses and Their Replication, FieldsVirology, 5th ed. Lippincott Williams and Wilkins; Tong et al., 2013,PLoS, pathogens, e1003657). In addition, antigenically distinct isolatescan also exist within the same subtype, referred to as drifted variants.IAVs exist mainly in the wild aquatic fowl reservoir (de Jong et al.,2007, J Virol, 81: 4315-4322; Taubenberger and Kash, 2010, Cell Host &Microbe, 7: 440-451; Webster et al., 1992, Microbiological Reviews, 56:152-179; Yoon et al., 2014, Current Topics in Microbiology andImmunology, 385: 359-375) and only a small number of mammalian hosts arecurrently recognized to sustain transmission and sustention of IAVs.

Canine influenza or dog flu is a common and contagious respiratorydisease of dogs caused by two IAVs: the H3N8 equine-origin influenzavirus that transferred to dogs in the United States around 1999(Crawford et al., 2005, Science, 310: 482-485); and the avian virus-likeH3N2 that transferred to dogs in Asia around 2005 (Song et al., 2008,Emerging Infectious Diseases, 14: 741-746). Recently, in 2015, anoutbreak of H3N2 canine influenza virus (CIV) similar to the onesdetected in dogs in Asia, was reported in the United States (2015,Javrna-J Am Vet med A, 246: 1049). Notably, H3N2 CIV seems to have abroad host range, as it has been isolated from cats during an outbreakof respiratory disease in a shelter in South Korea (Jeoung et al., 2013,Veterinary Microbiology, 165: 281-286; Song et al., 2011, The Journal ofGeneral Virology, 92: 2350-2355). CIV represents a new threat to caninehealth in the United States and worldwide, as the virus rapidly spreadsto dogs throughout the racing track circuit (Crawford et al., 2005,Science, 310: 482-485; Yoon et al., 2005, Emerging Infectious Diseases,11: 1974-1976) or animal shelters Crawford et al., 2005, Science, 310:482-485; Holt et al., 2010, Journal of the American Veterinary MedicalAssociation, 237: 71-73; Pecoraro et al., 2013, Journal of VeterinaryDiagnostic Investigation, 25: 402-406). CIV is a relatively new virusand almost all dogs are susceptible to infection when they are newlyexposed because they have not natural immunity. Most dogs that developCIV infection have a mild illness, but some dogs get very sick andrequire treatment (Gonzalez et al., 2014, J Virol, 88: 9208-9219). Therecent emergence of CIV has important implications, because theecological niche of IAVs has increased significantly and both of theseCIVs (H3N8 and H3N2) have continuously circulated in the dog populationsince they emerged, creating many opportunities for exposure in humansand other species. Importantly, as dogs are susceptible to mammalian(equine-origin H3N8 CIV) and avian (avian-origin H3N2 CIV) IAVs, theypossess all the attributes to become, like pigs, “mixing vessel” speciesfor the emergency of new IAVs with pandemic potential for humans. Thefact that dogs are the closest human companion animals makesreassortment between canine and human viruses more likely to occur. Infact, it has been shown that reassortments between H3N8 or H3N2 CIVs andhuman IAVs are feasible (Song et al., 2015, The Journal of GeneralVirology, 96: 254-258; Song et al., The Journal of General Virology, 93:551-554). Hence, society should be alert to the possible transmissionand potential emergence of CIVs in humans. This is particularlyalarming, because the introduction of novel, antigenically distinctglycoproteins (HA and NA) within the backbone of human IAVs haspreviously been associated with human pandemics (Yen et al., 2009,Current topics in microbiology and immunology, 333: 3-24).

Vaccination is universally accepted as the most effective strategy forthe prevention of influenza viral infections (Pica et al., 2013, AnnualReview of Medicine, 64: 189-202; Wong et al., 2013, ClinicalMicrobiology Reviews, 26: 476-492). To date, three types of influenzavirus vaccines have been approved by the United States FDA for humanuse: recombinant viral HA, inactivated influenza vaccines (IIVs), andlive attenuated influenza vaccines (LAIVs) (Pica et al., 2013, AnnualReview of Medicine, 64: 189-202; Belshe et al., 2007, The New EnglandJournal of Medicine, 356: 685-696; Cox et al., 2008, Influenza and otherRespiratory Viruses: 2: 211-219; Osterholm et al., 2012, The LancetInfectious Diseases, 12: 36-44; Pronker et al., 2012, Vaccine, 30:7344-7347). In dogs, IIV against H3N8 (and recently H3N2) CIVs arecommercially available. IIVs are administered intramuscularly and elicitprotective humoral immunity by inducing the production of neutralizingantibodies that target epitopes on HA, typically proximal to thereceptor binding site (Osterholm et al., 2012, The Lancet InfectiousDiseases, 12: 36-44; Belongia et al., 2009, Journal of InfectiousDiseases, 199: 159-167) that prevent (neutralize) viral infection. Onthe other hand, LAIV mimics the natural route of viral infection and areable to elicit more efficient cellular and humoral immune responses(Belshe et al., 2007, The New England Journal of Medicine, 356:685-696), providing better immunogenicity and protection against bothhomologous and heterologous influenza virus strains (Pica et al., 2013,Annual Review of Medicine, 64: 189-202; Gorse et al., 1991, Chest, 100:977-984).

In 2006, the American Veterinary Medical Association (AVMA) called forthe urgent development of an effective vaccine against CIV. A vaccinemade from inactivated virus have been developed that is administeredsubcutaneously as two doses to reduce the severity of the CIV diseaseand to reduce the incidence of CIV infection in naive dogs (Nobivac,Merck). However, to date, no LAIV for CIV infections has been developed.Thus there is a need in the art for improved vaccines for CIV. Thepresent invention satisfies this unmet need.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides an immunologicalcomposition comprising a live-attenuated canine influenza virus (LACIV),wherein the LACIV comprises one or more mutations in one or more of:segment 1 and segment 2 of the viral genome.

In one embodiment, the one or more mutations renders the LACIVtemperature sensitive such that the LACIV exhibits reduced viralreplication as compared to wildtype canine influenza virus at atemperature selected from the group consisting of normal bodytemperature and elevated body temperature.

In one embodiment, segment 1 comprises the nucleic acid sequence setforth in SEQ ID NO: 1. In one embodiment, segment 2 comprises thenucleic acid sequence set forth in SEQ ID NO: 2.

In one embodiment, the LACIV comprises one or more mutations in segment1, which encodes mutant PB2. In one embodiment, mutant PB2 comprises aN265S point mutation. In one embodiment, mutant PB2 comprises the aminoacid sequence set forth in SEQ ID NO: 3.

In one embodiment, the LACIV comprises one or more mutations in segment2, which encodes mutant PB1. In one embodiment, mutant PB1 comprises oneor more of: K391E point mutation, E581G point mutation, and A661T pointmutation. In one embodiment, mutant PB1 comprises a K391E pointmutation, a E581G point mutation, and a A661T point mutation. In oneembodiment, mutant PB1 comprises the amino acid sequence set forth inSEQ ID NO: 4.

In one embodiment, the LACIV comprises one or more mutations in segment1, which encodes mutant PB2; and one or more mutations in segment 2,which encodes mutant PB1.

In one embodiment, mutant PB2 comprises a N265S point mutation andmutant PB1 comprises a K391E point mutation, a E581G point mutation, anda A661T point mutation.

In one embodiment, the LACIV is derived from H3N8 subtype of influenza Avirus. In one embodiment, the LACIV expresses HA and NA of H3N8. In oneembodiment, the LACIV expresses HA and NA of H3N2.

In one embodiment, the composition is used for the treatment orprevention of canine influenza in a subject.

In one aspect, the method comprises a method for treating or preventingcanine influenza in a subject. The method comprises administering to thesubject an immunological composition comprising a live-attenuated canineinfluenza virus (LACIV), wherein the LACIV comprises one or moremutations in one or more of segment 1 and segment 2 of the viral genome.

In one embodiment, the one or more mutations renders the LACIVtemperature sensitive such that the LACIV exhibits reduced viralreplication as compared to wildtype canine influenza virus at atemperature selected from the group consisting of normal bodytemperature and elevated body temperature.

In one embodiment, segment 1 comprises the nucleic acid sequence setforth in SEQ ID NO: 1. In one embodiment, segment 2 comprises thenucleic acid sequence set forth in SEQ ID NO: 2.

In one embodiment, the LACIV comprises one or more mutations in segment1, which encodes mutant PB2. In one embodiment, mutant PB2 comprises aN265S point mutation. In one embodiment, mutant PB2 comprises the aminoacid sequence set forth in SEQ ID NO: 3.

In one embodiment, the LACIV comprises one or more mutations in segment2, which encodes mutant PB1. In one embodiment, mutant PB1 comprises oneor more of: K391E point mutation, E581G point mutation, and A661T pointmutation. In one embodiment, mutant PB1 comprises a K391E pointmutation, a E581G point mutation, and a A661T point mutation. In oneembodiment, mutant PB1 comprises the amino acid sequence set forth inSEQ ID NO: 4.

In one embodiment, the LACIV comprises one or more mutations in segment1, which encodes mutant PB2; and one or more mutations in segment 2,which encodes mutant PB1.

In one embodiment, mutant PB2 comprises a N265S point mutation andmutant PB1 comprises a K391E point mutation, a E581G point mutation, anda A661T point mutation.

In one embodiment, the LACIV is derived from H3N8 subtype of influenza Avirus. In one embodiment, the LACIV expresses HA and NA of H3N8. In oneembodiment, the LACIV expresses HA and NA of H3N2.

In one embodiment, the subject does not have canine influenza, andwherein the method induces immunity against one or more of: influenza Avirus subtype H3N8 and influenza A virus subtype H3N2. In oneembodiment, the subject is infected with at least one or more of:influenza A virus subtype H3N8 and influenza A virus subtype H3N2; andwherein the method induces a therapeutic immune response.

In one embodiment, the immunological composition is administeredintranasally, intratracheally, orally, intradermally, intramuscularly,intraperitoneally, intravenously, or subcutaneously.

In one embodiment, the subject is a dog.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of theinvention will be better understood when read in conjunction with theappended drawings. For the purpose of illustrating the invention, thereare shown in the drawings embodiments which are presently preferred. Itshould be understood, however, that the invention is not limited to theprecise arrangements and instrumentalities of the embodiments shown inthe drawings.

FIG. 1, comprising FIG. 1A and FIG. 1B, depicts the results ofexperiments demonstrating the effects of temperature on the activity ofLACIV viral polymerase. FIG. 1A: Schematic representation of segments 1(PB2) and 2 (PB1) of CIV WT (black) and LACIV (white). Amino acidsubstitutions N265S (PB2) and K391E, E581G, and A661T (PB1) to generatethe H3N8 LACIV are indicated. FIG. 1B: Minigenome activity. MDCK cells(12 well plate format, 3×10⁵ cells/well, triplicates) were transientlyco-transfected with 250 ng of ambisense pDZ expression plasmids encodingthe minimal requirements for viral genome replication and genetranscription (PB2, PB1, PA and NP), together with 500 ng of a vRNA-likeexpression plasmid encoding Gaussia luciferase (Gluc) under the controlof the canine polymerase I promoter (cpPol-I Gluc), and 100 ng of apCAGGS Cypridina luciferase (Cluc) plasmid to normalize transfectionefficiencies. After transfection, cells were placed at 33° C., 37° C. or39° C. and viral replication and transcription was evaluated 24 h laterby luminescence (Gluc). Gluc activity was normalized to that of Cluc.Data represent means and SD. Normalized reporter expression is relativeto that in the absence of pDZ NP plasmid. Data were represented asrelative activity considering WT H3N8 polymerase activity at eachtemperature as 100%. *, P<0.05 using a Student's t test.

FIG. 2, comprising FIG. 2A through FIG. 2C, depicts the results ofexperiments characterizing H3N8 LACIV in vitro: MDCK cells (12 wellplate format, 3×10⁵ cells/well, triplicates) were infected (MOI of0.001) with WT (black diamonds) and LACIV (gray squares) H3N8 CIVs andincubated at 33° C. (FIG. 2A), 37° C. (FIG. 2B) and 39° C. (FIG. 2C).TCS were collected at 12, 24, 48, 72, and 96 h p.i. and viral titerswere determined by immunofocus assay (FFU/ml). Data represent the meansand SD of the results determined in triplicate. Dotted lines indicatethe limit of detection (200 FFU/ml). *, P<0.05 using a Student's t test.

FIG. 3 depicts the results of experiments demonstrating the attenuationof H3N8 LACIV in vivo. Female 5-to-7-week-old C57BL/6 WT mice (N=6) wereinfected (i.n.) with 1×10⁵ PFU of WT or LACIV H3N8 CIVs. Three mice weresacrificed at days 2 (black) and 4 (gray) p.i. and lungs were harvestedfor virus titrations using an immunofocus assay (FFU/ml). Data representthe means and SD. Dotted line indicate limit of detection (200 FFU/ml).ND, virus not detected.

FIG. 4, comprising FIG. 4A and FIG. 4B, depicts the results ofexperiments demonstrating the immunogenicity and protection efficacy ofH3N8 LACIV against homologous viral challenge: Female 5-to-7-week-oldC57BL/6 WT mice (N=6) were vaccinated (i.n.) with 1×10³ PFU of WT orLACIV H3N8 CIVs. Mice mock (PBS) vaccinated or vaccinated (i.m.) with100 μl of an H3N8 CIV IIV (Nobivac) were used as internal controls. FIG.4A) Induction of humoral responses: At 14 days post-vaccination, micewere bled and sera was evaluated for the presence of total IgGantibodies against H3N8 CIV proteins using cell extracts ofMDCK-infected cells by ELISA. MDCK mock-infected cell extracts were usedto evaluate the specificity of the antibody response. OD, opticaldensity. Data represent the means+/−SD of the results for 6 individualmice. * (Nobivac vs LACIV), ** (WT vs LACIV) or *** (WT vs Nobivac),P<0.05 using a Student's t test. FIG. 4B) Protection efficacy: At 15days post-vaccination, same mice were challenged (i.n.) with 1×10⁵ PFUof H3N8Wt CIV. To evaluate viral replication, mice were euthanized atdays 2 (N=3) and 4 (N=3) post-challenge and lungs were harvested,homogenized, and used to quantify viral titers by immunofocus assay(FFU/ml). The dotted line indicates the limit of detection (200 FFU/ml).ND, virus not detected. Data represent the means+/−SDs. *, P<0.05 usinga Student's t test.

FIG. 5, comprising FIG. 5A through FIG. 5D, depicts the results ofexperiments investigating the ex vivo infection of canine trachealexplants with A/Canine/NY/2009 wild-type (WT) and LAIV (LACIV). (FIG.5A) Histological features of dog tracheas infected with 200 plaqueforming units (PFU) of WT and LACIV or mock-infected with infectionmedia. Lesions are shown in sections stained with haematoxylin and eosin(H&E) at the indicated days post-infection. (FIG. 5B) Infected cellswere detected by immunohistochemical staining of the viral NP. Positivecells are stained in brown. Black horizontal bars represent 20 μm. (FIG.5C) Graphical representation of bead clearance assays in infected andcontrol dog tracheal explants. Lines represent the average time to clearthe beads in three independent experiments. Error bars represent SEM.(FIG. 5D) Growth kinetics of WT and LACIV in canine tracheal explants.Vertical bars represent average from three independent experiments.

FIG. 6, comprising FIG. 6A and FIG. 6B, depicts the results ofexperiments demonstrating the immunogenicity and protection efficacy ofH3N8 LACIV against heterologous H3N2 CIV challenge: Female5-to-7-week-old C57BL/6 WT mice were vaccinated (i.n.) with 1×10³ PFU ofWT and LACIV H3N8 CIVs. Mice mock (PBS) vaccinated or vaccinated (i.m.)with 100 μl of the H3N8 (Noviback) and an H3N2 CIV IIV were used asinternal controls. FIG. 6A) Antibody cross-reactivity against theheterologous CIV H3N2: At 14 days post-vaccination, mice were bled andsera was evaluated by ELISA for total IgG antibodies against H3N2 CIVproteins using cell extracts of MDCK-infected cells. Mock-infected MDCKcell extracts were used to evaluate the specificity of the antibodyresponse. OD, optical density. Data represent the means+/−SD of theresults for 6 individual mice. * (Nobivac vs LACIV), ** (WT vs LACIV) or*** (WT vs Nobivac), P<0.05 using a Student's t test. FIG. 6B)Protection efficacy of H3N8 LACIV against heterologous H3N2 CIVchallenge: At 15 days post-vaccination, mice were challenged (i.n.) with1×10⁵ PFU of WT H3N2 CIV. To evaluate WT H3N2 CIV replication, mice weresacrificed at days 2 (N=3) and 4 (N=3) post-challenge and lungs wereharvested, homogenized, and used to evaluate the presence of virus byimmunofocus assay (FFU/ml). The dotted line indicates the limit ofdetection (200 FFU/ml). ND, virus not detected. Data represent themeans+/−SDs. *, P<0.05 using a Student's t test.

FIG. 7 is a schematic representation of the generation of CIV H3N2 LAIV:Amino acid substitutions N265S (PB2) and K391E, E581G, and A661T (PB1)were introduced into the A/canine/NY/dog23/2009 H3N8 (CIV H3N8) togenerate the CIV H3N8 LAIV. CIV H3N8 LAIV was used as a master donorvirus (MDV) to generate the CIV H3N2 LAIV that contains the internalviral segments (PB2, PB1, PA, NP, M and NS) of CIV H3N8 LAIV and the HAand NA of A/Ca/IL/41915/2015 H3N2 (CIV H3N2).

FIG. 8, comprising FIG. 8A through FIG. 8C, depicts the results ofexperiments examining the multicycle growth kinetics of CIV H3N2 LAIV:Canine MDCK cells (12-well plate format, 5×10⁵ cells/well, triplicates)were infected at low multiplicity of infection (MOI, 0.001) withA/Canine/Illinois/11613/2015 H3N2 (CIV H3N2 WT), A/Canine/NY/Dog23/2009H3N8 (CIV H3N8 WT) and the two LAIVs (CIV H3N2 LAIV and CIV H3N8 LAIV)and incubated at 33° C. (FIG. 8A), 37° C. (FIG. 8B) and 39° C. (FIG.8C). Tissue culture supernatants were collected at 12, 24, 48, 72 and 96hours post-infection. Viral titers in tissue culture supernatants weredetermined by immunofocus assay (Focus Forming Units, FFU/ml) using ananti-NP monoclonal antibody (HT-103). Data represent the means+/−SDs ofthe results determined in triplicate. Dotted black lines indicates thelimit of detection (200 FFU/ml).

FIG. 9, comprising FIG. 9A and FIG. 9B, depicts the results ofexperiments demonstrating the attenuation of CIV H3N2 LAIV: Female6-to-8-week-old C57BL/6 WT mice (N=6) were infected intranasally (i.n.)with 1×10⁵ FFU of CIV H3N2 WT or CIV H3N2 LAIV. Presence of viruses inthe lungs (FIG. 9A) and the nasal mucosal (FIG. 9B) of infected micewere evaluated at days 2 (N=3) and 4 (N=3) post-infection by immunofocusassay (FFU/ml) using an anti-NP monoclonal antibody (HT-103). Datarepresent the means+/−SDs. Dotted black lines indicate limit ofdetection (200 FFU/ml). *, P<0.05 and **, P<0.001 (WT vs LAIV) usingStudent's t test (n=3).

FIG. 10, comprising FIG. 10A and FIG. 10B, depicts the results ofexperiments investigating the induction of humoral responses by CIV H3N2LAIV: Female 6-to-8-week-old C57BL/6 WT mice (N=6) were immunized with1×10³ FFU of CIV H3N2 WT or CIV H3N2 LAIV. Mice were also mockvaccinated or vaccinated with 100 ul/mice of an inactivated CIV H3N2vaccine (Zoetis) as negative and positive controls, respectively. At 14days post-vaccination, mice were bled and the sera were collected andevaluated individually by ELISA for IgG antibodies against totalinfluenza virus protein using cell extracts of MDCK cells infected withA/Canine/Illinois/11613/2015 H3N2 WT virus (FIG. 10A) orA/Canine/NY/Dog23/2009 H3N8 WT virus (FIG. 10B). Mock-infected cellextracts were used to evaluate the specificity of the antibody response.OD, optical density. Data represent the means+/−SDs of the results for 6individual mice.

FIG. 11, comprising FIG. 11A and FIG. 11B, depicts the results ofexperiments investigating the protection efficacy of CIV H3N2 LAIV:Female 6-to-8-week-old C57BL/6 WT mice (N=12) were vaccinated with 1×10³FFU of CIV H3N2 WT or CIV H3N2 LAIV. Mice were also mock vaccinated orvaccinated with 100 ul/mice of a CIV H3N2 inactivated vaccine (Zoetis)as negative and positive controls, respectively. Two weekspost-vaccination, mice (N=6) were challenged with 1×10⁵ FFU of CIV H3N2WT (FIG. 11A) or CIV H3N8 WT (FIG. 11B). Viral titers of challengedviruses at days 2 (N=3) and 4 (N=3) post-infection were evaluated fromlung homogenates by immunofocus assay (FFU/ml) using an anti-NPmonoclonal (HT-103 or HB-65 respectively). Dotted black lines indicatelimit of detection (200 FFU/ml). Data represent the means+/−SDs.

FIG. 12 is a schematic representation of bivalent CIV LAIV comprisingthe CIV H3N8 LAIV and the CIV H3N2 LAIV.

FIG. 13, comprising FIG. 13A and FIG. 13B, depict the nucleotidesequence of mutant segment 1 (FIG. 13A) and the amino acid sequence ofmutant PB2 (FIG. 13B) of H3N8 LACIV, derived from A/Canine/NY/Dog23/2009H3N8. The nucleotide changes resulting in the N265S amino acid changeare in bold and underlined in FIG. 13A. The N265S amino acid change isin bold and underlined in FIG. 13B.

FIG. 14, comprising FIG. 14A and FIG. 14B, depict the nucleotidesequence of mutant segment 2 (FIG. 14A) and the amino acid sequence ofmutant PB1 (FIG. 14B) of H3N8 LACIV, derived from A/Canine/NY/Dog23/2009H3N8. The nucleotide changes resulting in the K391E, E581G, and A661Tamino acid changes are in bold and underlined in FIG. 14A. The K391E,E581G, and A661T amino acid changes are in bold and underlined in FIG.14B.

DETAILED DESCRIPTION

The present invention relates to compositions and methods for thetreatment and prevention of canine influenza virus (CIV) and CIV-relatedpathology. The present invention is based in part upon the discoverythat various mutations in segment 1 and segment 2 of the CIV genome,thereby encoding mutant PB2 and PB1 protein, render the virus to betemperature-sensitive. For example, it is described herein that suchmutations result in CIV exhibiting reduced viral replication at normaland elevated body temperature as compared to wildtype CIV. However, thetemperature-sensitive CIV is able to induce a CIV-specific immuneresponse. Thus, the temperature-sensitive CIV described herein is alive-attenuated canine influenza vaccine (LACIV). Importantly, the LACIVinduces a greater CIV-specific immune response as compared to aninactivated CIV vaccine.

In certain embodiments, the present invention provides a composition forthe treatment and prevention of canine influenza virus (CIV) andCIV-related pathology. In one embodiment, the composition comprises aLACIV having one or more mutations in segment 1 and/or segment 2 of theviral genome. For example, in one embodiment, the LACIV encodes mutantPB2 and/or mutant PB1. In certain embodiments, mutant PB2 comprises aN265S point mutation. In certain embodiments, mutant PB1 comprises atleast one of a K391E point mutation, a E581G point mutation, or A661Tpoint mutation.

In certain embodiments, the present invention provides a compositioncomprising a master donor virus (MDV) having one or more mutations insegment 1 and/or segment 2 of the viral genome. In one embodiment, theMDV comprises mutant H3N8 segment 1 and/or segment 2, as describedherein. In certain embodiments, the MDV can be used to generate an LACIVwhich is protective against other pathogens. For example, in certainembodiments, an LACIV against another influenza strain can be generatedby using the MDV to express one or more viral proteins of the otherstrain.

In certain embodiments, the present invention provides a method fortreating or preventing CIV and CIV-related pathology, comprisingadministering a composition comprising a LACIV. In certain embodiments,the method comprises intranasal delivery of the LACIV.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described.

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value,as such variations are appropriate to perform the disclosed methods.

The term “antibody,” as used herein, refers to an immunoglobulinmolecule which specifically binds with an antigen. Antibodies can beintact immunoglobulins derived from natural sources or from recombinantsources and can be immunoreactive portions of intact immunoglobulins.The antibodies in the present invention may exist in a variety of formsincluding, for example, polyclonal antibodies, monoclonal antibodies,Fv, Fab and F(ab)₂, as well as single chain antibodies and humanizedantibodies (Harlow et al., 1999, In: Using Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989,In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houstonet al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al.,1988, Science 242:423-426).

The term “antigen” or “Ag” as used herein is defined as a molecule thatprovokes an immune response. This immune response may involve eitherantibody production, or the activation of specificimmunologically-competent cells, or both. The skilled artisan willunderstand that any macromolecule, including virtually all proteins orpeptides, can serve as an antigen. Furthermore, antigens can be derivedfrom recombinant or genomic DNA. A skilled artisan will understand thatany DNA, which comprises a nucleotide sequences or a partial nucleotidesequence encoding a protein that elicits an immune response thereforeencodes an “antigen” as that term is used herein. Furthermore, oneskilled in the art will understand that an antigen need not be encodedsolely by a full length nucleotide sequence of a gene. It is readilyapparent that the present invention includes, but is not limited to, theuse of partial nucleotide sequences of more than one gene and that thesenucleotide sequences are arranged in various combinations to elicit thedesired immune response. Moreover, a skilled artisan will understandthat an antigen need not be encoded by a “gene” at all. It is readilyapparent that an antigen can be generated synthesized or can be derivedfrom a biological sample.

As used herein, the term “autologous” is meant to refer to any materialderived from the same individual to which it is later to bere-introduced into the individual.

As used herein, by “combination therapy” is meant that a first agent isadministered in conjunction with another agent. “In conjunction with”refers to administration of one treatment modality in addition toanother treatment modality. As such, “in conjunction with” refers toadministration of one treatment modality before, during, or afterdelivery of the other treatment modality to the individual. Suchcombinations are considered to be part of a single treatment regimen orregime.

As used herein, the term “concurrent administration” means that theadministration of the first therapy and that of a second therapy in acombination therapy overlap with each other.

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated thenthe animal's health continues to deteriorate. In contrast, a “disorder”in an animal is a state of health in which the animal is able tomaintain homeostasis, but in which the animal's state of health is lessfavorable than it would be in the absence of the disorder. Leftuntreated, a disorder does not necessarily cause a further decrease inthe animal's state of health.

An “effective amount” as used herein, means an amount which provides atherapeutic or prophylactic benefit.

The term “expression” as used herein is defined as the transcriptionand/or translation of a particular nucleotide sequence driven by itspromoter.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, such as cosmids, plasmids (e.g., naked or contained in liposomes)and viruses (e.g., lentiviruses, retroviruses, adenoviruses, andadeno-associated viruses) that incorporate the recombinantpolynucleotide.

“Homologous” refers to the sequence similarity or sequence identitybetween two polypeptides or between two nucleic acid molecules. When aposition in both of the two compared sequences is occupied by the samebase or amino acid monomer subunit, e.g., if a position in each of twoDNA molecules is occupied by adenine, then the molecules are homologousat that position. The percent of homology between two sequences is afunction of the number of matching or homologous positions shared by thetwo sequences divided by the number of positions compared×100. Forexample, if 6 of 10 of the positions in two sequences are matched orhomologous then the two sequences are 60% homologous. By way of example,the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, acomparison is made when two sequences are aligned to give maximumhomology.

The term “immunoglobulin” or “Ig,” as used herein, is defined as a classof proteins, which function as antibodies. Antibodies expressed by Bcells are sometimes referred to as the BCR (B cell receptor) or antigenreceptor. The five members included in this class of proteins are IgA,IgG, IgM, IgD, and IgE. IgA is the primary antibody that is present inbody secretions, such as saliva, tears, breast milk, gastrointestinalsecretions and mucus secretions of the respiratory and genitourinarytracts. IgG is the most common circulating antibody. IgM is the mainimmunoglobulin produced in the primary immune response in most subjects.It is the most efficient immunoglobulin in agglutination, complementfixation, and other antibody responses, and is important in defenseagainst bacteria and viruses. IgD is the immunoglobulin that has noknown antibody function, but may serve as an antigen receptor. IgE isthe immunoglobulin that mediates immediate hypersensitivity by causingrelease of mediators from mast cells and basophils upon exposure toallergen.

As used herein, the term “immune response” includes T-cell mediatedand/or B-cell mediated immune responses. Exemplary immune responsesinclude T cell responses, e.g., cytokine production and cellularcytotoxicity, and B cell responses, e.g., antibody production. Inaddition, the term immune response includes immune responses that areindirectly affected by T cell activation, e.g., antibody production(humoral responses) and activation of cytokine responsive cells, e.g.,macrophages. Immune cells involved in the immune response includelymphocytes, such as B cells and T cells (CD4+, CD8+, Th1 and Th2cells); antigen presenting cells (e.g., professional antigen presentingcells such as dendritic cells, macrophages, B lymphocytes, Langerhanscells, and non-professional antigen presenting cells such askeratinocytes, endothelial cells, astrocytes, fibroblasts,oligodendrocytes); natural killer cells; myeloid cells, such asmacrophages, eosinophils, mast cells, basophils, and granulocytes.

“Isolated” means altered or removed from the natural state. For example,a nucleic acid or a peptide naturally present in a living animal is not“isolated,” but the same nucleic acid or peptide partially or completelyseparated from the coexisting materials of its natural state is“isolated.” An isolated nucleic acid or protein can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a host cell.

“Parenteral” administration of an immunogenic composition includes,e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), orintrasternal injection, or infusion techniques.

The terms “patient,” “subject,” “individual,” and the like are usedinterchangeably herein, and refer to any animal, or cells thereofwhether in vitro or in situ, amenable to the methods described herein.In certain non-limiting embodiments, the patient, subject or individualis a human.

The term “simultaneous administration,” as used herein, means that afirst therapy and second therapy in a combination therapy areadministered with a time separation of no more than about 15 minutes,such as no more than about any of 10, 5, or 1 minutes. When the firstand second therapies are administered simultaneously, the first andsecond therapies may be contained in the same composition (e.g., acomposition comprising both a first and second therapy) or in separatecompositions (e.g., a first therapy in one composition and a secondtherapy is contained in another composition).

By the term “specifically binds,” as used herein with respect to anantibody, is meant an antibody which recognizes a specific antigen, butdoes not substantially recognize or bind other molecules in a sample.For example, an antibody that specifically binds to an antigen from onespecies may also bind to that antigen from one or more species. But,such cross-species reactivity does not itself alter the classificationof an antibody as specific. In another example, an antibody thatspecifically binds to an antigen may also bind to different allelicforms of the antigen. However, such cross reactivity does not itselfalter the classification of an antibody as specific. In some instances,the terms “specific binding” or “specifically binding,” can be used inreference to the interaction of an antibody, a protein, or a peptidewith a second chemical species, to mean that the interaction isdependent upon the presence of a particular structure (e.g., anantigenic determinant or epitope) on the chemical species; for example,an antibody recognizes and binds to a specific protein structure ratherthan to proteins generally. If an antibody is specific for epitope “A,”the presence of a molecule containing epitope A (or free, unlabeled A),in a reaction containing labeled “A” and the antibody, will reduce theamount of labeled A bound to the antibody.

The term “normal temperature” or “normal body temperature” as usedherein refers to the temperature of a healthy subject. For example, incertain instances the “normal body temperature” in a human subject is inthe range of about 36° C. to about 38° C. In certain instances, in acanine subject, “normal body temperature” is in the range of about 38°C. to about 39.5° C.

The term “elevated temperature” or “elevated body temperature” as usedherein refers to a temperature in a subject that is greater than the“normal body temperature” of a subject of a given organism. In certaininstances “elevated body temperature” may be indicative of a fever,infection, or other illness. In certain instances, elevated bodytemperature in a human subject is greater than about 37° C. In certaininstances, elevated body temperature in a canine subject is greater thanabout 38.5° C.

The term “therapeutic” as used herein means a treatment and/orprophylaxis. A therapeutic effect is obtained by suppression, remission,or eradication of a disease state.

The term “therapeutically effective amount” refers to the amount of thesubject compound that will elicit the biological or medical response ofa tissue, system, or subject that is being sought by the researcher,veterinarian, medical doctor or other clinician. The term“therapeutically effective amount” includes that amount of a compoundthat, when administered, is sufficient to prevent development of, oralleviate to some extent, one or more of the signs or symptoms of thedisorder or disease being treated. The therapeutically effective amountwill vary depending on the compound, the disease and its severity andthe age, weight, etc., of the subject to be treated.

To “treat” a disease as the term is used herein, means to reduce thefrequency or severity of at least one sign or symptom of a disease ordisorder experienced by a subject.

The term “transfected” or “transformed” or “transduced” as used hereinrefers to a process by which exogenous nucleic acid is transferred orintroduced into the host cell. A “transfected” or “transformed” or“transduced” cell is one which has been transfected, transformed ortransduced with exogenous nucleic acid. The cell includes the primarysubject cell and its progeny.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

DESCRIPTION

The present invention provides immunological compositions and methodsuseful for the inhibition, prevention and treatment of canine influenzaand canine influenza related diseases and disorders. In one embodiment,the immunological composition comprises a live-attenuated virus (LAV).

In one embodiment, the present invention provides atemperature-sensitive LAV of a canine influenza virus. For example, itis demonstrated herein that one or more mutations in segment 1 and/orsegment 2 of the CIV genome renders the virus to betemperature-sensitive. The temperature-sensitive LACIV of the presentinvention exhibits reduced viral replication, as compared to wildtypeCIV, at both normal body temperature and at elevated or fevertemperatures. However, the temperature sensitive LACIV providesantigen-specific immune responses and protection against CIV. In oneembodiment, the LACIV provides at least the same antigen-specific immuneresponses and protection against CIV compared to wildtype CIV. Incertain embodiments, the LACIV provides greater antigen-specific immuneresponses and protection against CIV as compared to inactivated CIV.

In general, wild-type influenza viruses contain a segmented genome with8 segments as described in Table 1 below:

TABLE 1 Segment Gene Product 1 PB2 (Polymerase (basic) protein 2) 2 PB1(Polymerase (basic) protein 1) 3 PA (Polymerase (acidic) protein) 4 HA(Hemagglutinin) 5 NP (Nucleoprotein) 6 NA (Neuraminidase) 7 M1 (Matrixprotein 1) and M2 (Matrix protein 2) 8 NS1 (non-structural protein 1)and NEP/NS2 (non-structural protein 2)

In certain embodiments, the present invention provides an immunologicalcomposition comprising segment 1 and/or segment 2, wherein segment 1and/or segment 2 comprise one or more mutations. For example, in certainembodiments, the immunological composition comprises an LAV, comprisingone or more mutations in segment 1 and/or segment 2. In one embodiment,the immunological composition comprises a LACIV, comprising one or moremutations in segment 1 and/or segment 2.

The present invention also provides methods of preventing, inhibiting,and treating CIV and CIV-related diseases and disorders. In oneembodiment, the methods of the invention induce immunity against CIV bygenerating an immune response directed to CIV. In one embodiment, themethods of the invention induce production of CIV-specific antibodies.In one embodiment, the methods of the invention prevent CIV-relatedpathology. In one embodiment, the methods of the invention compriseadministering an immunological composition comprising a LAV, wherein theLAV comprises one or more mutations in segment 1 and/or segment 2, to asubject in need thereof. In one embodiment, the methods compriseadministering an immunological composition to a subject in need thereof,thereby inducing immunity to CIV.

Compositions

The present invention provides immunological compositions that whenadministered to a subject in need thereof, elicit an immune responsedirected against canine influenza virus (CIV). In some embodiments, thecomposition includes polypeptides, nucleotides, vectors, or vaccines.Further, when the compositions are administered to a subject, theyelicit an immune response that serves to protect the inoculated subjectagainst canine influenza. As exemplified herein, the composition can beobtained in large quantities for use as a vaccine.

In one embodiment, the present invention provides compositions that areuseful as immunomodulatory agents, for example, in stimulating immuneresponses and in preventing canine influenza and canineinfluenza-related pathology.

Live-attenuated viruses can be used as immunostimulatory agents toinduce the production of CIV-specific antibodies and protect againstcanine influenza and canine influenza-related pathology. Therefore, inone embodiment, the composition of the invention comprises alive-attenuated CIV (LACIV), wherein the LACIV comprises one or moremutations in the viral genome to render the LACIV temperature sensitive.For example, in one embodiment, the LACIV comprises one or moremutations in segment 1 of the viral genome. The one or more mutations insegment 1 of the viral genome encode a mutant PB2 protein. In oneembodiment, the LACIV comprises one or more mutations in segment 2 ofthe viral genome. The one or more mutations in segment 2 of the viralgenome encode a mutant PB1 protein. In one embodiment, the LACIVcomprises one or more mutations in segment 1 and one or more mutationsin segment 2.

In one embodiment, the LACIV is based upon the genome of InfluenzaA/canine/NY/dog23/2009 H3N8. Wildtype nucleic acid sequences for eachsegment of Influenza A/canine/NY/dog23/2009 H3N8 and wildtype amino acidsequences for the encoded proteins are summarized in Table 2 below:

TABLE 2 Wildtype sequences for Influenza A/canine/NY/dog23/2009 H3N8Segments Gene Products Segment 1 (SEQ ID NO: 5) PB2 (SEQ ID NO: 6)Segment 2 (SEQ ID NO: 7) PB1 (SEQ ID NO: 8) Segment 3 (SEQ ID NO: 9) PA(SEQ ID NO: 10) Segment 4 (SEQ ID NO: HA (SEQ ID NO: 12) 11) Segment 5(SEQ ID NO: NP (SEQ ID NO: 14) 13) Segment 6 (SEQ ID NO: NA (SEQ ID NO:16) 15) Segment 7 (SEQ ID NO: M1 (SEQ ID NO: 18) M2 (SEQ ID 17) NO: 19)Segment 8 (SEQ ID NO: NS1 (SEQ ID NO: NEP/NS2 (SEQ ID 20) 21) NO: 22)

In one embodiment, the composition comprises one or more mutations inthe nucleic acid sequences of segment 1, encoding PB2, and/or segment 2,encoding PB1. Thus, in certain embodiments, the composition encodesmutant PB1 and/or mutant PB2. As described herein, the one or moremutations renders the virus to be temperature-sensitive, exhibitedreduced viral replication at normal or elevated temperatures.

In some embodiments, the invention provides a composition comprising oneor more mutations in segment 1. For example, in one embodiment, thecomposition comprises segment 1 having one or more mutation whichresults in the production of mutant PB2 having a point mutation at aminoacid residue 265. For example, in one embodiment, the mutant PB2comprises the amino acid sequence of SEQ ID NO: 6, except having a pointmutation at amino acid residue 265. For example, in one embodiment, themutant PB2 comprises a N265S point mutation, where the mutant PB2comprises a serine at amino acid residue 265.

In one embodiment, the composition comprises a nucleic acid sequenceencoding a mutant PB2 having an amino acid sequence of SEQ ID NO: 3. Inone embodiment, the composition comprises a nucleic acid sequenceencoding a mutant PB2 that is substantially homologous to SEQ ID NO: 3.For example, in certain embodiments, the composition comprises a nucleicacid sequence that encodes a mutant PB2 that is at least 50% homologous,at least 60% homologous, at least 70% homologous, at least 80%homologous, at least 90% homologous, at least 95% homologous, at least98% homologous, at least 99% homologous, or at least 99.5% homologous toSEQ ID NO: 3. In one embodiment, the composition comprises a nucleicacid sequence encoding a mutant PB2 that is substantially homologous toSEQ ID NO: 3, where mutant PB2 that is substantially homologous to SEQID NO: 3 comprises the N265S point mutation.

In one embodiment, the composition comprises a mutant segment 1comprising the nucleotide sequence of SEQ ID NO: 1. In one embodiment,the composition comprises nucleotide sequence that is substantiallyhomologous to SEQ ID NO: 1. For example, in certain embodiments, thecomposition comprises a nucleotide sequence that is at least 50%homologous, at least 60% homologous, at least 70% homologous, at least80% homologous, at least 90% homologous, at least 95% homologous, atleast 98% homologous, at least 99% homologous, or at least 99.5%homologous to SEQ ID NO: 1. In one embodiment, the composition comprisesa nucleotide sequence that is substantially homologous to SEQ ID NO: 1,where the mutant PB2 encoded by the nucleotide sequence that issubstantially homologous to SEQ ID NO: 1 comprises the N265S pointmutation.

In some embodiments, the invention provides a composition comprising oneor more mutations in segment 2. For example, in one embodiment, thecomposition comprises segment 2 having one or more mutation whichresults in the production of mutant PB1 having a point mutation at oneor more of: amino acid residue 391, amino acid residue 581, and aminoacid residue 661. For example, in one embodiment, the mutant PB2comprises the amino acid sequence of SEQ ID NO: 8, except having a pointmutation at one or more of: amino acid residue 391, amino acid residue581, and amino acid residue 661. For example, in one embodiment, themutant PB1 comprises a K391E point mutation, where the mutant PB1comprises a glutamic acid at amino acid residue 391. In one embodiment,the mutant PB1 comprises a E581G point mutation, where the mutant PB1comprises a glycine at amino acid residue 581. In one embodiment, themutant PB1 comprises a A661T point mutation, where the mutant PB1comprises a threonine at amino acid residue 661.

In one embodiment, the composition comprises a nucleic acid sequenceencoding a mutant PB1 having an amino acid sequence of SEQ ID NO: 4. Inone embodiment, the composition comprises a nucleic acid sequenceencoding a mutant PB1 that is substantially homologous to SEQ ID NO: 4.For example, in certain embodiments, the composition comprises a nucleicacid sequence that encodes a mutant PB1 that is at least 50% homologous,at least 60% homologous, at least 70% homologous, at least 80%homologous, at least 90% homologous, at least 95% homologous, at least98% homologous, at least 99% homologous, or at least 99.5% homologous toSEQ ID NO: 4. In one embodiment, the composition comprises a nucleicacid sequence encoding a mutant PB1 that is substantially homologous toSEQ ID NO: 4, where mutant PB1 that is substantially homologous to SEQID NO: 4 comprises one or more of the K391E point mutation, E581G pointmutation, and A661T point mutation.

In one embodiment, the composition comprises a mutant segment 2comprising the nucleotide sequence of SEQ ID NO: 2. In one embodiment,the composition comprises nucleotide sequence that is substantiallyhomologous to SEQ ID NO: 2. For example, in certain embodiments, thecomposition comprises a nucleotide sequence that is at least 50%homologous, at least 60% homologous, at least 70% homologous, at least80% homologous, at least 90% homologous, at least 95% homologous, atleast 98% homologous, at least 99% homologous, or at least 99.5%homologous to SEQ ID NO: 2. In one embodiment, the composition comprisesa nucleotide sequence that is substantially homologous to SEQ ID NO: 2,where the mutant PB1 encoded by the nucleotide sequence that issubstantially homologous to SEQ ID NO: 2 comprises one or more of theK391E point mutation, E581G point mutation, and A661T point mutation.

In certain embodiments, the composition comprises one or more mutationsin segment 1 and one or more mutations in segment 2. For example, incertain embodiments, the composition comprises segment 1 having a N265Spoint mutation, and segment 2 having one or more of K391E pointmutation, E581G point mutation, and A661T point mutation.

In certain embodiments, the composition comprises one or more mutationsin the nucleic acid sequences of segment 1 and/or segment 2, whilecomprising wildtype nucleic acid sequences for the rest of the segmentedgenome. For example, in one embodiment, the LACIV comprises one or moremutations in segment 1 and comprises wildtype segment 2, segment 3,segment 4, segment 5, segment 6, segment 7, and segment 8. In oneembodiment, the LACIV comprises one or more mutation is segment 2 andcomprises wildtype segment 1, segment 3, segment 4, segment 5, segment6, segment 7, and segment 8. In one embodiment, the LACIV comprises oneor more mutations in segment 1 and segment 2 and comprises wildtypesegment 3, segment 4, segment 5, segment 6, segment 7, and segment 8.

In certain embodiments, the composition comprises one or more mutationsin segment 1 and/or segment 2, in combination with one or more mutationsin one or more other segments of the viral genome.

For example, in one embodiment, the composition further comprises one ormore mutations in segment 8. In one embodiment, the compositioncomprises a deletion mutant of segment 8, such that the coding region ofNS1 protein is truncated or deleted, as described in PCT PatentApplication PCT/US2016/047711, filed on Aug. 19, 2016, claiming priorityto U.S. Provisional Patent Application No. 62/207,576, each of whichapplications are incorporated by reference in their entirety.

For example, in one embodiment, the composition further comprises one ormore mutations in segment 4. In one embodiment, the compositioncomprises a deletion mutant of segment 4, such that HA is not expressed,as described in PCT Patent Application PCT/US2016/047726, filed on Aug.19, 2016, claiming priority to U.S. Provisional Patent Application No.62/207,579, each of which applications are incorporated by reference intheir entirety.

In certain embodiments, the composition comprises a mutant segment 1,mutant segment 2, or combination thereof, as described herein, incombination with one or more nucleotide sequences encoding anotherantigen. For example, in certain embodiments, the composition comprisesa mutant segment 1, mutant segment 2, or combination thereof, asdescribed herein, in combination with one or more nucleotide sequencesencoding one or more antigens of another virus or strain. For example,in certain aspects, the H3N8 LACIV described herein, comprising a mutantsegment 1, mutant segment 2, or combination thereof can be used as amaster donor virus (MDV). For example, an MDV comprising an H3N8comprising a mutant segment 1, mutant segment 2, or combination thereofdescribed herein, can be modified to comprise one or more nucleotidesequences encoding one or more of PB2, PB1, PA, NP, HA, NA, M1, M2, NS1,or NEP/NS2 from another influenza strain. As such a compositioncomprising an H3N8 comprising a mutant segment 1, mutant segment 2, orcombination thereof described herein can provide protection against adifferent strain, when the composition expresses an antigen of thedifferent strain. For example, in one embodiment, a compositioncomprises the backbone of a H3N8 LACIV comprising a mutant segment 1,mutant segment 2, or combination thereof described herein, furthercomprising one or more nucleotide sequences encoding one or more of PB2,PB1, PA, NP, HA, NA, M1, M2, NS1, or NEP/NS2 from another influenzastrain. In one embodiment, the composition comprises the backbone of aH3N8 LACIV comprising a mutant segment 1, mutant segment 2, orcombination thereof described herein, further comprising one or morenucleotide sequences encoding one or more of HA or NA of a differentinfluenza strain, including but not limited to H3N2 CIV. For example,the composition comprising the backbone of a H3N8 LACIV describedherein, may be modified to express one or more viral proteins of a newlyemergent strain, thereby providing protection against the newly emergentstrain.

In one embodiment, the composition comprises segment 1, segment 2,segment 3, segment 5, segment 7, and segment 8 of H3N8 LACIV, describedherein, comprising one or more point mutations in one or more of segment1 and segment 2, where the composition further comprises segment 4 andsegment 6, of a different CIV strain, including but not limited to H3N2CIV.

In one embodiment, the composition comprises a mutant segment 1 of H3N8,mutant segment 2 of H3N8, or a combination thereof, further comprisingsegment 4, segment 6, or a combination thereof of H3N2. In oneembodiment, the composition comprising a mutant segment 1 of H3N8,mutant segment 2 of H3N8, or a combination thereof, further comprisingsegment 4, segment 6, or a combination thereof of H3N2 is an H3N2 LACIV.In certain aspects, the mutant segment 1, mutant segment 2, orcombination thereof of H3N8 provides for the temperature sensitiveattenuated phenotype of the LACIV, while the segment 4, segment 6, orcombination thereof, of H3N2, encodes H3N2 HA, H3N2 NA, or combinationthereof to elicit an H3N2 specific immune response in the subject.

The nucleotide sequence of segment 4 of A/Canine/Illinois/11613/2015H3N2 is provided in SEQ ID NO: 23. The amino acid sequence of HA,encoded by segment 4, of A/Canine/Illinois/11613/2015 H3N2 is providedin SEQ ID NO: 24. The nucleotide sequence of segment 6 ofA/Canine/Illinois/11613/2015 H3N2 is provided in SEQ ID NO: 25. Theamino acid sequence of NA, encoded by segment 6, ofA/Canine/Illinois/11613/2015 H3N2 is provided in SEQ ID NO: 26.

In one embodiment, the composition comprises a nucleic acid sequenceencoding HA having the amino acid sequence of SEQ ID NO: 24. In oneembodiment, the composition comprises a nucleic acid sequence encodingHA that is substantially homologous to SEQ ID NO: 24. For example, incertain embodiments, the composition comprises a nucleic acid sequencethat encodes HA that is at least 50% homologous, at least 60%homologous, at least 70% homologous, at least 80% homologous, at least90% homologous, at least 95% homologous, at least 98% homologous, atleast 99% homologous, or at least 99.5% homologous to SEQ ID NO: 24.

In one embodiment, the complication comprises a segment 4 comprising thenucleotide sequence of SEQ ID NO: 23. In one embodiment, the compositioncomprises nucleotide sequence that is substantially homologous to SEQ IDNO: 23. For example, in certain embodiments, the composition comprises anucleotide sequence that is at least 50% homologous, at least 60%homologous, at least 70% homologous, at least 80% homologous, at least90% homologous, at least 95% homologous, at least 98% homologous, atleast 99% homologous, or at least 99.5% homologous to SEQ ID NO: 23.

In one embodiment, the composition comprises a nucleic acid sequenceencoding NA having the amino acid sequence of SEQ ID NO: 26. In oneembodiment, the composition comprises a nucleic acid sequence encodingHA that is substantially homologous to SEQ ID NO: 26. For example, incertain embodiments, the composition comprises a nucleic acid sequencethat encodes HA that is at least 50% homologous, at least 60%homologous, at least 70% homologous, at least 80% homologous, at least90% homologous, at least 95% homologous, at least 98% homologous, atleast 99% homologous, or at least 99.5% homologous to SEQ ID NO:

26.

In one embodiment, the complication comprises a segment 6 comprising thenucleotide sequence of SEQ ID NO: 25. In one embodiment, the compositioncomprises nucleotide sequence that is substantially homologous to SEQ IDNO: 25. For example, in certain embodiments, the composition comprises anucleotide sequence that is at least 50% homologous, at least 60%homologous, at least 70% homologous, at least 80% homologous, at least90% homologous, at least 95% homologous, at least 98% homologous, atleast 99% homologous, or at least 99.5% homologous to SEQ ID NO: 25.

In one embodiment, the composition comprises a plurality of LACIVdescribed herein. For example, in one embodiment, the compositioncomprises a first LACIV, comprising mutant segment 1, mutant segment 2,or combination thereof of H3N8, where the first LACIV comprises segment4, segment 6, or a combination thereof of H3N8; and the compositionfurther comprises a second LACIV, comprising mutant segment 1, mutantsegment 2, or combination thereof of H3N8, where the second LACIVcomprises segment 4, segment 6, or a combination thereof of H3N2. Incertain embodiments, the composition induces an immune response againstboth H3N8 and H3N2 CIV.

In certain embodiments, the composition comprises a polynucleotideencoding mutant PB2 and/or mutant PB1. The polynucleotide can be RNA orDNA. In one embodiment, the composition comprises a DNA vaccine.

The nucleic acid sequences include both the DNA sequence that istranscribed into RNA and the RNA sequence that is translated into apolypeptide. According to other embodiments, the polynucleotides of theinvention are inferred from the amino acid sequence of the polypeptidesof the invention. As is known in the art several alternativepolynucleotides are possible due to redundant codons, while retainingthe biological activity of the translated polypeptides.

Further, the invention encompasses an isolated nucleic acid comprising anucleotide sequence having substantial homology to a nucleotide sequenceof an isolated nucleic acid encoding a polypeptide disclosed herein.Preferably, the nucleotide sequence of an isolated nucleic acid encodinga polypeptide of the invention is “substantially homologous,” that is,is about 60% homologous, more preferably about 70% homologous, even morepreferably about 80% homologous, more preferably about 90% homologous,even more preferably, about 95% homologous, and even more preferablyabout 99% homologous to a nucleotide sequence of an isolated nucleicacid encoding a polypeptide of the invention.

It is to be understood explicitly that the scope of the presentinvention encompasses homologs, analogs, variants, fragments,derivatives and salts, including shorter and longer polypeptides andpolynucleotides, as well as polypeptide and polynucleotide analogs withone or more amino acid or nucleic acid substitution, as well as aminoacid or nucleic acid derivatives, non-natural amino or nucleic acids andsynthetic amino or nucleic acids as are known in the art, with thestipulation that these modifications must preserve the immunologicactivity of the original molecule. Specifically any active fragments ofthe active polypeptides as well as extensions, conjugates and mixturesare included and are disclosed herein according to the principles of thepresent invention.

The invention should be construed to include any and all isolatednucleic acids which are homologous to the nucleic acids described andreferenced herein, provided these homologous nucleic acids encodepolypeptides having the biological activity of the polypeptidesdisclosed herein.

The skilled artisan would understand that the nucleic acids of theinvention encompass a RNA or a DNA sequence encoding a polypeptide ofthe invention, and any modified forms thereof, including chemicalmodifications of the DNA or RNA which render the nucleotide sequencemore stable when it is cell free or when it is associated with a cell.Chemical modifications of nucleotides may also be used to enhance theefficiency with which a nucleotide sequence is taken up by a cell or theefficiency with which it is expressed in a cell. Any and allcombinations of modifications of the nucleotide sequences arecontemplated in the present invention.

Further, any number of procedures may be used for the generation ofmutant, derivative or variant forms of a protein of the invention usingrecombinant DNA methodology well known in the art such as, for example,that described in Sambrook et al. (2012, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory, New York), and in Ausubel et al.(1997, Current Protocols in Molecular Biology, John Wiley & Sons, NewYork). Procedures for the introduction of amino acid changes in apolypeptide or polypeptide by altering the DNA sequence encoding thepolypeptide are well known in the art and are also described in these,and other, treatises.

According to yet another embodiment, composition of the invention,comprising the nucleic acid sequences or combination of nucleic acidsequences of the present invention, is capable of generating aCIV-specific immune response. In another embodiment, the composition ofthe invention, comprising the nucleic acid sequences or combination ofnucleic acid sequences of the present invention, is capable ofgenerating CIV-specific antibodies. In certain embodiments, thecomposition is able to protect against CIV, including H3N8 CIV and H3N2CIV.

In one embodiment, the composition of the invention comprises apolypeptide, or a fragment of a polypeptide, a homolog, a variant, aderivative or a salt of a polypeptide having the sequence of any one ormore of SEQ ID NO: 3 and SEQ ID NO: 4.

The invention should also be construed to include any form of apolypeptide having substantial homology to the polypeptides disclosedherein. Preferably, a polypeptide which is “substantially homologous” isabout 50% homologous, more preferably about 70% homologous, even morepreferably about 80% homologous, more preferably about 90% homologous,even more preferably, about 95% homologous, and even more preferablyabout 99% homologous to amino acid sequence of the polypeptidesdisclosed herein.

According to yet another embodiment, composition of the invention,comprising the polypeptide or combination of polypeptides of the presentinvention, is capable of generating a CIV-specific immune response. Inanother embodiment, the composition of the invention, comprising thepolypeptide or combination of polypeptides of the present invention, iscapable of generating CIV-specific antibodies. In certain embodiments,the composition is able to protect against CIV, including H3N8 CIV andH3N2 CIV.

The present invention should also be construed to encompass “mutants,”“derivatives,” and “variants” of the polypeptides of the invention (orof the DNA encoding the same) which mutants, derivatives and variantsare polypeptides which are altered in one or more amino acids (or, whenreferring to the nucleotide sequence encoding the same, are altered inone or more base pairs) such that the resulting polypeptide (or DNA) isnot identical to the sequences recited herein, but has the samebiological property as the polypeptides disclosed herein.

Live Attenuated Virus (LAV)

The invention relates in part to the generation, selection andidentification of live attenuated viruses (LAV) that generate aCIV-specific immune response, and the use of such viruses in vaccine andpharmaceutical formulations.

As described herein, in certain embodiments the LACIV comprises one ormore mutations in segment 1 and/or one or more mutations in segment 2that render the virus to be temperature-sensitive. For example, in oneembodiment, the temperature-sensitive LACIV exhibits reduced viralreplication at normal and elevated temperatures. However, thetemperature-sensitive LACIV induces CIV-specific immune responses andantibody production, and is thus able to protect against CIV andCIV-related pathology.

Any mutant virus or strain which has at least one mutation can beselected and used in accordance with the invention. In one embodiment,naturally occurring mutants or variants, or spontaneous mutants can beselected that include at least one mutation in segment 1 and/or segment2, as described elsewhere herein. In another embodiment, mutant virusescan be generated by exposing the virus to mutagens, such as ultravioletirradiation or chemical mutagens, or by multiple passages and/or passagein non-permissive hosts. Screening in a differential growth system canbe used to select for those mutants having at least one mutation insegment 1 and/or segment 2, as described elsewhere herein. For viruseswith segmented genomes, the attenuated phenotype can be transferred toanother strain having a desired antigen by reassortment, (i.e., bycoinfection of the attenuated virus and the desired strain, andselection for reassortants displaying both phenotypes).

In another embodiment, mutations can be engineered into an influenzavirus, including, but not limited to H3N8 CIV or H3N2 CIV using “reversegenetics” approaches. In this way, natural or other mutations whichconfer the attenuated phenotype can be engineered into vaccine strains.For example, deletions, insertions, or substitutions of the codingregion of segment 1, encoding PB2, and/or segment 2, encoding PB1 can beengineered. Deletions, substitutions or insertions in the non-codingregion of segment 1 and/or segment 2 are also contemplated. To this end,mutations in the signals responsible for the transcription, replication,polyadenylation and/or packaging of segment 1 and/or segment 2 can beengineered.

In certain instances, the reverse genetics technique involves thepreparation of synthetic recombinant viral RNAs that contain thenon-coding regions of the negative strand virus RNA which are essentialfor the recognition by viral polymerases and for packaging signalsnecessary to generate a mature virion. The recombinant RNAs aresynthesized from a recombinant DNA template and reconstituted in vitrowith purified viral polymerase complex to form recombinantribonucleoproteins (RNPs) which can be used to transfect cells. In someinstances, a more efficient transfection is achieved if the viralpolymerase proteins are present during transcription of the syntheticRNAs either in vitro or in vivo. The synthetic recombinant RNPs can berescued into infectious virus particles. The foregoing techniques aredescribed in U.S. Pat. No. 5,166,057 issued Nov. 24, 1992; in U.S. Pat.No. 5,854,037 issued Dec. 29, 1998; in European Patent Publication EP0702085A1, published Feb. 20, 1996; in U.S. patent application Ser. No.09/152,845; in International Patent Publications PCT WO97/12032published Apr. 3, 1997; WO96/34625 published Nov. 7, 1996; in EuropeanPatent Publication EP-A780475; WO 99/02657 published Jan. 21, 1999; WO98/53078 published Nov. 26, 1998; WO 98/02530 published Jan. 22, 1998;WO 99/15672 published Apr. 1, 1999; WO 98/13501 published Apr. 2, 1998;WO 97/06270 published Feb. 20, 1997; and EPO 780 47SA1 published Jun.25, 1997, each of which is incorporated by reference herein in itsentirety.

Attenuated viruses generated by the reverse genetics approach can beused in the vaccine and pharmaceutical formulations described herein.Reverse genetics techniques can also be used to engineer additionalmutations to other viral genes important for vaccine production—i.e.,the epitopes of useful vaccine strain variants can be engineered intothe attenuated virus. Alternatively, completely foreign epitopes,including antigens derived from other viral or non-viral pathogens canbe engineered into the attenuated strain.

In an alternate embodiment, a combination of reverse genetics techniquesand reassortant techniques can be used to engineer attenuated viruseshaving the desired epitopes. For example, an attenuated virus (generatedby natural selection, mutagenesis or by reverse genetics techniques) anda strain carrying the desired vaccine epitope (generated by naturalselection, mutagenesis or by reverse genetics techniques) can beco-infected in hosts that permit reassortment of the segmented genomes.Reassortants that display both the attenuated phenotype and the desiredepitope can then be selected.

The attenuated virus of the present invention can itself be used as theactive ingredient in vaccine or pharmaceutical formulations. In certainembodiments, the attenuated virus can be used as the vector or“backbone” of recombinantly produced vaccines. To this end, the “reversegenetics” technique can be used to engineer mutations or introduceforeign epitopes into the attenuated virus, which would serve as the“parental” strain. In this way, vaccines can be designed forimmunization against strain variants, or in the alternative, againstcompletely different infectious agents or disease antigens.

For example, in one embodiment, the immunological composition of theinvention comprises a live attenuated virus, engineered to express oneor more epitopes or antigens of CIV along with epitopes or antigens ofanother pathogen. For example, the attenuated virus can be engineered toexpress neutralizing epitopes of other preselected strains.Alternatively, epitopes of other viruses can be built into theattenuated mutant virus. Alternatively, epitopes of non-viral infectiouspathogens (e.g., parasites, bacteria, fungi) can be engineered into thevirus.

In one embodiment, the attenuated viruses selected for use in theinvention is capable of inducing a robust anti-CIV response in thehost—a feature which contributes to the generation of a strong immuneresponse when used as a vaccine, and which has other biologicalconsequences that make the viruses useful as pharmaceutical agents forthe prevention and/or treatment of other viral infections, or otherdiseases.

The attenuated viruses, which induce a CIV-specific immune response inhosts, may also be used in pharmaceutical formulations for theprophylaxis or treatment of other influenza infections, orinfluenza-related pathology. In this regard, the tropism of theattenuated virus can be altered to target the virus to a desired targetorgan, tissue or cells in vivo or ex vivo. Using this approach, theCIV-specific immune response can be induced locally, at the target site,thus avoiding or minimizing the side effects of systemic treatments. Tothis end, the attenuated virus can be engineered to express a ligandspecific for a receptor of the target organ, tissue or cells.

Vaccine

In certain aspects, the immunological composition is useful as avaccine, where the immunological composition induces an immune responseto the antigen in a cell, tissue or mammal. Preferably, the vaccineinduces a protective immune response in the mammal. As used herein, an“immunological composition” may comprise, by way of examples, alive-attenuated virus (LAV), an antigen (e.g., a polypeptide), a nucleicacid encoding an antigen (e.g., an antigen expression vector), or a cellexpressing or presenting an antigen or cellular component. In particularembodiments the immunological composition comprises or encodes all orpart of any polypeptide antigen described herein, or an immunologicallyfunctional equivalent thereof. In other embodiments, the immunologicalcomposition is in a mixture that comprises an additionalimmunostimulatory agent or nucleic acids encoding such an agent.Immunostimulatory agents include but are not limited to an additionalantigen, an immunomodulator, an antigen presenting cell or an adjuvant.In other embodiments, one or more of the additional agent(s) iscovalently bonded to the antigen or an immunostimulatory agent, in anycombination. In certain embodiments, the antigenic composition isconjugated to or comprises an HLA anchor motif amino acids.

In the context of the present invention, the term “vaccine” refers to asubstance that induces anti-CIV immunity or suppresses CIV uponinoculation into an animal.

The invention encompasses vaccine formulations comprising liveattenuated virus (LAV), wherein the LAV is a live attenuated canineinfluenza virus (LACIV). For example, in certain embodiments, the LACIVis temperature-sensitive, exhibiting reduced viral replication at normaland elevated temperatures, as compared to wildtype CIV. In oneembodiment, the vaccine comprises a LACIV comprising one or moremutations in segment 1 and/or segment 2, and a suitable excipient. Thevirus used in the vaccine formulation may be selected from naturallyoccurring mutants or variants, mutagenized viruses or geneticallyengineered viruses. Attenuated strains of CIV can also be generated viareassortment techniques, or by using a combination of the reversegenetics approach and reassortment techniques. Naturally occurringvariants include viruses isolated from nature as well as spontaneousoccurring variants generated during virus propagation. The attenuatedvirus can itself be used as the active ingredient in the vaccineformulation. Alternatively, the attenuated virus can be used as thevector or “backbone” of recombinantly produced vaccines. To this end,recombinant techniques such as reverse genetics (or, for segmentedviruses, combinations of the reverse genetics and reassortmenttechniques) may be used to engineer mutations or introduce foreignantigens into the attenuated virus used in the vaccine formulation. Inthis way, vaccines can be designed for immunization against strainvariants, or in the alternative, against completely different infectiousagents or disease antigens.

In one embodiment, the vaccine formulation comprises a plurality ofmutant CIV. In one embodiment, the vaccine formulation comprises abivalent vaccine comprising H3N8 LACIV, described herein, in combinationwith H3N2 LACIV, where the H3N2 LACIV is based upon the H3N8 LACIVbackbone but engineered to express H3N2 HA and NA viral proteins (seeExample 2).

In one embodiment, the vaccine formulation may comprise one or more ofthe LACIV, described herein, in combination with other mutant CIV thatinduce an anti-CIV immune response. For example, in one embodiment, thevaccine formulation comprises a single cycle infectious CIV having oneor more mutations in segment 4, such that HA is not expressed. In oneembodiment, the vaccine formulation comprises a mutant CIV comprising adeletion mutant in segment 8.

In one embodiment, the present invention comprises a method ofgenerating a LACIV, comprising contacting a host cell with apolynucleotide comprising the nucleic acid sequences of segment 1 and/orsegment 2, having one or more mutations, described elsewhere herein.

Propagation of the virus in culture is known to persons in the art.Briefly, the virus is grown in the media compositions in which the hostcell is commonly cultured. Suitable host cells for the replication ofCIV include, e.g., Vero cells, BHK cells, MDCK cells, 293 cells COScells, and CEK cells, including 293T cells, COST cells. Commonly,co-cultures including two of the above cell lines, e.g., MDCK cells andeither 293T or COS cells are employed at a ratio, e.g., of 1:1, toimprove replication efficiency. Typically, cells are cultured in astandard commercial culture medium, such as Dulbecco's modified Eagle'smedium supplemented with serum (e.g., 10% fetal bovine serum), or inserum free medium, under controlled humidity and CO₂ concentrationsuitable for maintaining neutral buffered pH (e.g., at pH between 7.0and 7.2). Optionally, the medium contains antibiotics to preventbacterial growth, e.g., penicillin, streptomycin, etc., and/oradditional nutrients, such as L-glutamine, sodium pyruvate,non-essential amino acids, additional supplements to promote favorablegrowth characteristics, e.g., trypsin, β-mercaptoethanol, and the like.

Procedures for maintaining mammalian cells in culture have beenextensively reported, and are known to those of skill in the art.General protocols are provided, e.g., in Freshney (1983) Culture ofAnimal Cells: Manual of Basic Technique, Alan R. Liss, New York; Paul(1975) Cell and Tissue Culture, 5th ed., Livingston, Edinburgh; Adams(1980) Laboratory Techniques in Biochemistry and Molecular Biology-CellCulture for Biochemists, Work and Burdon (eds.) Elsevier, Amsterdam.Additional details regarding tissue culture procedures of particularinterest in the production of influenza virus in vitro include, e.g.,Merten et al. (1996) Production of influenza virus in cell cultures forvaccine preparation. In Cohen and Shafferman (eds) Novel Strategies inDesign and Production of Vaccines, which is incorporated herein in itsentirety. Additionally, variations in such procedures adapted to thepresent invention are readily determined through routineexperimentation.

Cells for production of a virus can be cultured in serum-containing orserum free medium. In some case, e.g., for the preparation of purifiedviruses, it is desirable to grow the host cells in serum freeconditions. Cells can be cultured in small scale, e.g., less than 25 mlmedium, culture tubes or flasks or in large flasks with agitation, inrotator bottles, or on microcarrier beads (e.g., DEAE-Dextranmicrocarrier beads, such as Dormacell, Pfeifer & Langen; Superbead, FlowLaboratories; styrene copolymer-tri-methylamine beads, such as Hillex,SoloHill, Ann Arbor) in flasks, bottles or reactor cultures.Microcarrier beads are small spheres (in the range of 100-200 microns indiameter) that provide a large surface area for adherent cell growth pervolume of cell culture. For example a single liter of medium can includemore than 20 million microcarrier beads providing greater than 8000square centimeters of growth surface. For commercial production ofviruses, e.g., for vaccine production, it is often desirable to culturethe cells in a bioreactor or fermenter. Bioreactors are available involumes from under 1 liter to in excess of 100 liters, e.g., Cyto3Bioreactor (Osmonics, Minnetonka, Minn.); NBS bioreactors (New BrunswickScientific, Edison, N.J.); laboratory and commercial scale bioreactorsfrom B. Braun Biotech International (B. Braun Biotech, Melsungen,Germany).

Virtually any heterologous gene sequence may be constructed into theviruses of the invention for use in vaccines. Preferably, epitopes thatinduce a protective immune response to any of a variety of pathogens, orantigens that bind neutralizing antibodies may be expressed by or aspart of the viruses. For example, heterologous gene sequences that canbe constructed into the viruses of the invention for use in vaccinesinclude but are not limited to epitopes of human immunodeficiency virus(HIV) such as gp120; hepatitis B virus surface antigen (HBsAg); theglycoproteins of herpes virus (e.g. gD, gE); VP1 of poliovirus;antigenic determinants of non-viral pathogens such as bacteria andparasites, to name but a few. In another embodiment, all or portions ofimmunoglobulin genes may be expressed. For example, variable regions ofanti-idiotypic immunoglobulins that mimic such epitopes may beconstructed into the viruses of the invention. In yet anotherembodiment, tumor associated antigens may be expressed.

Either a live recombinant viral vaccine or an inactivated recombinantviral vaccine can be formulated. A live vaccine may be preferred becausemultiplication in the host leads to a prolonged stimulus of similar kindand magnitude to that occurring in natural infections, and therefore,confers substantial, long-lasting immunity. Production of such liverecombinant virus vaccine formulations may be accomplished usingconventional methods involving propagation of the virus in cell cultureor in the allantois of the chick embryo followed by purification.

Many methods may be used to introduce the vaccine formulations describedabove, these include but are not limited to introduction intranasally,intratracheally, orally, intradermally, intramuscularly,intraperitoneally, intravenously, and subcutaneously. It may bepreferable to introduce the virus vaccine formulation via the naturalroute of infection of the pathogen for which the vaccine is designed, orvia the natural route of infection of the parental attenuated virus.

A vaccine of the present invention, comprising a LACIV, could beadministered once. Alternatively, a vaccine of the present invention,comprising a LACIV, could be administered twice or three or more timeswith a suitable interval between doses. Alternatively, a vaccine of thepresent invention, comprising a LACIV, could be administered as often asneeded to an animal, preferably a mammal.

Methods

The invention provides a method for treating or preventing canineinfluenza infection or a CIV-related disease or disorder. In oneembodiment, the method comprises administering an immunologicalcomposition comprising a live-attenuated virus (LAV), wherein the LAV isa LACIV. In one embodiment, the method comprises administering animmunological composition comprising an LACIV comprising one or moremutations in segment 1 and/or segment 2, to a subject in need thereof.

As described herein, in certain embodiments, the LACIV is temperaturesensitive, exhibiting decreased viral replication at normal and elevatedtemperatures, as compared to wildtype CIV. For example, in certainembodiments, the viral replication of LACIV is 2-fold less, 3-fold less,5-fold less, 10-fold less, 15-fold less, 20-fold less, 50-fold less,100-fold less, 500-fold less, or 1000-fold less, than wild type CIV atnormal or elevated body temperature.

In certain embodiments, the LACIV induces an enhanced immune response ascompared to an inactivated CIV. For example, in certain embodiments, theinduced immune response of LACIV is 2-fold more, 3-fold more, 5-foldmore, 10-fold more, 15-fold more, 20-fold more, 50-fold more, 100-foldmore, 500-fold more, or 1000-fold more, than inactivated CIV. The immuneresponse induced the LACIV can be measured using standard assays. Forexample, in certain embodiments, the immune response induced by LACIV ismeasured by detecting the amount of CIV-specific antibodies produced inthe subject following administration of LACIV.

The therapeutic compositions of the invention may be administeredprophylactically or therapeutically to subjects suffering from, or atrisk of, or susceptible to, developing the disease or condition. Suchsubjects may be identified using standard clinical methods. In thecontext of the present invention, prophylactic administration occursprior to the manifestation of overt clinical symptoms of disease, suchthat a disease or disorder is prevented or alternatively delayed in itsprogression. In the context of the field of medicine, the term “prevent”encompasses any activity which reduces the burden of mortality ormorbidity from disease. Prevention can occur at primary, secondary andtertiary prevention levels. While primary prevention avoids thedevelopment of a disease, secondary and tertiary levels of preventionencompass activities aimed at preventing the progression of a diseaseand the emergence of symptoms as well as reducing the negative impact ofan already established disease by restoring function and reducingdisease-related complications.

In certain embodiments, the subject is a mammal. For example, thesubject may include, but is not limited to, a human, primate, cow,horse, sheep, pig, dog, cat, or rodent. In one embodiment, the subjectis a dog. The method may be used to treat or prevent CIV or CIV-relatedpathology in any breed or species of dog. In certain embodiments, therelative amount of active ingredient in a single dose, or the frequencyof doses, will vary depending on the age, sex, weight, or breed ofsubject (e.g. dog).

The composition may be combined with an adjuvant. An adjuvant refers toa compound that enhances the immune response when administered together(or successively) with the immunological composition. Examples ofsuitable adjuvants include cholera toxin, salmonella toxin, alum andsuch, but are not limited thereto. Furthermore, a vaccine of thisinvention may be combined appropriately with a pharmaceuticallyacceptable carrier. Examples of such carriers are sterilized water,physiological saline, phosphate buffer, culture fluid and such.Furthermore, the vaccine may contain as necessary, stabilizers,suspensions, preservatives, surfactants and such. The vaccine isadministered systemically or locally. Vaccine administration may beperformed by single administration or boosted by multipleadministrations.

Administration

In one embodiment, the methods of the present invention compriseadministering an immunological composition of the invention directly toa subject in need thereof. Administration of the composition cancomprise, for example, intranasal, intramuscular, intravenous,peritoneal, subcutaneous, intradermal, as well as topicaladministration.

Furthermore, the actual dose and schedule can vary depending on whetherthe compositions are administered in combination with otherpharmaceutical compositions, or depending on inter-individualdifferences in pharmacokinetics, drug disposition, and metabolism. Oneskilled in the art can easily make any necessary adjustments inaccordance with the exigencies of the particular situation.

Pharmaceutical Compositions

The present invention envisions treating or preventing CIV orCIV-related pathology in a mammal by the administration of a therapeuticcomposition of the invention to a mammal in need thereof. Administrationof the composition in accordance with the present invention may becontinuous or intermittent, depending, for example, upon the recipient'sphysiological condition, whether the purpose of the administration istherapeutic or prophylactic, and other factors known to skilledpractitioners. The administration of the compositions of the inventionmay be essentially continuous over a preselected period of time or maybe in a series of spaced doses. Both local and systemic administrationis contemplated. The amount administered will vary depending on variousfactors including, but not limited to, the composition chosen, theparticular disease, the weight, the physical condition, and the age ofthe mammal, and whether prevention or treatment is to be achieved. Suchfactors can be readily determined by the clinician employing animalmodels or other test systems which are well known to the art.

The present invention encompasses pharmaceutical compositions comprisinga LACIV to be used as anti-viral agents or as agents against CIV-relateddiseases and disorders. The pharmaceutical compositions have utility asan anti-viral prophylactic and may be administered to a subject at riskof getting infected or is expected to be exposed to a virus. Forexample, subjects traveling to parts of the world where CIV is prevalentcan be administered a pharmaceutical composition of the invention. Incertain embodiments, subjects who are expected to be in contact withother subjects at risk, can be administered a pharmaceutical compositionof the invention.

The LACIV of the invention may be engineered using the methods describedherein to express proteins or peptides which would target the viruses toa particular site. In one embodiment, where the site to be targetedexpresses a receptor to a growth factor, e.g., VEGF, EGF, or PDGF, theLACIV may be engineered to express the appropriate growth factor orportion(s) thereof. Thus, in accordance with the invention, the LACIVmay be engineered to express any target gene product, includingpeptides, proteins, such as enzymes, hormones, growth factors, antigensor antibodies, which will function to target the virus to a site in needof anti-viral, antibacterial, anti-microbial or anti-cancer activity.

Methods of introduction include but are not limited to intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, and oral routes. The pharmaceutical compositions of thepresent invention may be administered by any convenient route, forexample by infusion or bolus injection, by absorption through epithelialor mucocutaneous linings (e.g., oral mucosa, rectal and intestinalmucosa, etc.) and may be administered together with other biologicallyactive agents. Administration can be systemic or local. In addition, ina preferred embodiment it may be desirable to introduce thepharmaceutical compositions of the invention into the lungs by anysuitable route. Pulmonary administration can also be employed, e.g., byuse of an inhaler or nebulizer, and formulation with an aerosolizingagent.

In a specific embodiment, it may be desirable to administer thepharmaceutical compositions of the invention locally to the area in needof treatment; this may be achieved by, for example, and not by way oflimitation, local infusion during surgery, topical application, e.g., inconjunction with a wound dressing after surgery, by injection, by meansof a catheter, by means of a suppository, or by means of an implant,said implant being of a porous, non-porous, or gelatinous material,including membranes, such as sialastic membranes, or fibers.

In certain embodiments, the pharmaceutical composition is a veterinarypharmaceutical composition suitable for administration to a veterinarysubject, including but not limited to a canine subject. Exemplary caninesubjects include dogs, wolves, foxes, coyotes, and jackals.

In certain embodiments, the veterinary pharmaceutical composition is“palatable,” meaning an oral veterinary composition that is readilyaccepted by canines, including dogs, without any coaxing or with somecoaxing. Palatable compositions are compositions that score at least 2using a palatability assessment method wherein dog owners score thecomposition from 0 to 3, wherein dogs scoring 0 do not consume thecomposition; dogs scoring 1 consume the composition after some time;dogs scoring 2 consume the composition with some coaxing and dogsscoring 3 consume the composition readily. A skilled person iswell-versed in these palatability standards and scoring regimes. Inanother embodiment, the daily dose for dogs may be around 100 mg/kg.Veterinary pharmaceutical agents that may be included in thecompositions of the invention are well-known in the art (see e.g. Plumb'Veterinary Drug Handbook, 5th Edition, ed. Donald C. Plumb, BlackwellPublishing, (2005) or The Merck Veterinary Manual, 9th Edition, (January2005)).

In yet another embodiment, the pharmaceutical composition can bedelivered in a controlled release system. In one embodiment, a pump maybe used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng.14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N.Engl. J. Med. 321:574). In another embodiment, polymeric materials canbe used (see Medical Applications of Controlled Release, Langer and Wise(eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled DrugBioavailability, Drug Product Design and Performance, Smolen and Ball(eds.), Wiley, New York (1984); Ranger & Peppas, 1983, J. Macromol. Sci.Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science 228:190;During et al., 1989, Ann. Neurol. 25:351 (1989); Howard et al., 1989, J.Neurosurg. 71:105). In yet another embodiment, a controlled releasesystem can be placed in proximity of the composition's target, i.e., thelung, thus requiring only a fraction of the systemic dose (see, e.g.,Goodson, 1984, in Medical Applications of Controlled Release, supra,vol. 2, pp. 115-138). Other controlled release systems are discussed inthe review by Langer (1990, Science 249:1527-1533).

The pharmaceutical compositions of the present invention comprise atherapeutically effective amount of the attenuated virus, and apharmaceutically acceptable carrier. In a specific embodiment, the term“pharmaceutically acceptable” means approved by a regulatory agency ofthe Federal or a state government or listed in the U.S. Pharmacopeia orother generally recognized pharmacopeiae for use in animals, and moreparticularly in humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the pharmaceuticalcomposition is administered. Saline solutions and aqueous dextrose andglycerol solutions can also be employed as liquid carriers, particularlyfor injectable solutions. Suitable pharmaceutical excipients includestarch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,silica gel, sodium stearate, glycerol monostearate, talc, sodiumchloride, dried skim milk, glycerol, propylene, glycol, water and thelike. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. These compositions can be formulated as asuppository. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa therapeutically effective amount of the Therapeutic, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the patient. Theformulation should suit the mode of administration.

The amount of the pharmaceutical composition of the invention which willbe effective in the treatment or prevention of a particular disease ordisorder will depend on the nature of the disease or disorder, and canbe determined by standard clinical techniques. In addition, in vitroassays may optionally be employed to help identify optimal dosageranges. The precise dose to be employed in the formulation will alsodepend on the route of administration, and the seriousness of thedisease or disorder, and should be decided according to the judgment ofthe practitioner and each patient's circumstances. Effective doses maybe extrapolated from dose-response curves derived from in vitro oranimal model test systems.

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the present invention andpractice the claimed methods. The following working examples therefore,specifically point out the preferred embodiments of the presentinvention, and are not to be construed as limiting in any way theremainder of the disclosure.

Example 1: A Temperature Sensitive Live-Attenuated Canine InfluenzaVirus H3N8 Vaccine

It has been reported in recent years the emergence of two influenza Avirus (IAV) subtypes in dogs, the canine influenza virus (CIV) H3N8 andH3N2 of equine and avian origin, respectively. Vaccination serves as thebest therapeutic option to protect against influenza viral infections.To date, only inactivate influenza vaccines (IIVs) are available for thetreatment of CIV infections in dogs. However, the efficacy of currentcanine IIVs is suboptimal, and novel approaches are necessary for theprevention of disease caused by this contagious canine respiratorypathogen.

IAV is a respiratory pathogen that, at least in humans, is limited tothe cooler (33° C.) upper respiratory tract and leads to pathology viareplication in the warmer (37° C.) lower respiratory tract (Maassab.,1968, Nature, 219:645-646). The temperature gradient between these twoareas in the respiratory tract enabled the development of cold-adapted(ca), temperature-sensitive (ts), attenuated (att) viruses thatreplicates in the cooler upper respiratory tract (33° C.) to trigger animmune response but cannot damage the warmer lower respiratory tract(37° C.) due to the elevated temperatures restricting replication. Thisca, ts, att signature has been mapped to five amino acid residueslocated in three viral proteins of A/Ann Arbor/6/60 H2N2 (A/AA/6/60):the polymerase basic 2 (PB2; N265S), the polymerase basic 1 (PB1; K391E,D581G, and A661T) and the nucleoprotein (NP; D34G) (Cox et al., 1988,Virology, 167:554-567, Snyder et al., 1988, J Virol, 62:488-495). Themechanism of attenuation is not fully understood but most likelyinvolves multiple steps in the replication cycle of the virus (Chan etal., 2008, Virology, 380:304-311). Importantly, when the ca signature ofA/AA/6/60 was introduced into influenza A/Puerto Rico/8/34 H1N1 (PR8) orA/California/04/09 H1N1 (pH1N1) viruses, a similar ts phenotype of theseviruses was showed in tissue culture cells and in validated mice modelsof influenza infections (Cox et al., 2015, J Virol, 89(6): 3421-3426,Jin et al., 2004, J Virol, 78:995-998, Zhou et al., 2012, Vaccine, 30:3691-3702).

Reported herein is the generation of a recombinant, temperaturesensitive H3N8 CIV for its implementation as a live attenuated influenzavaccine (LAIV) candidate. In order to develop a LAIV for the treatmentof CIV H3N8 infections, we introduced the four ts, ca, att mutationspresent in A/AA/6/60 LAIV into the CIV H3N8 (referred to henceforth asLACIV) and rescued this virus using plasmid-based reverse geneticstechniques (Martinez-Sobrido et al., 2010, Journal of visualizedexperiments, 42; doi: 10.3791/2057). Introduction of the ts, ca, attmutations of A/AA/6/60 into the backbone of H3N8 CIV resulted in a virusthat efficiently replicate in vitro at lower (33° C.), important forvaccine production, but not at higher (37° C. and 39° C.) temperatures,demonstrating that the LAIV mutations of the current human LAIV are ableto confer a ts phenotype to the H3N8 CIV. Importantly, the H3N8 LACIVwas safe and able to confer, upon a single intranasal immunization dose,protective immune responses against homologous challenge with H3N8 CIV.Notably, protection conferred by our H3N8 LACIV is more efficient thanthat provided with currently available H3N8 CIV IIV, representing abetter option for the control of CIV in the dog population.

The materials and methods employed in these experiments are nowdescribed.

Cells and Viruses

Human embryonic kidney 293T cells (293T; ATCC CRL-11268) and Madin-Darbycanine kidney cells (MDCK; ATCC CCL-34) were grown at 37° C. with 5%CO₂, in Dulbecco's modified Eagle's medium (DMEM; Mediatech, Inc.)supplemented with 10% fetal bovine serum (FBS), and 1% PSG (penicillin,100 units/ml; streptomycin 100 μg/ml; L-glutamine, 2 mM) (Nogales etal., 2014, J Virol, 88: 10525-10540).

Recombinant wild-type (WT) and live-attenuated (LACIV) H3N8 CIVs weregenerated using A/canine/NY/dog23/2009 H3N8 plasmid-based reversegenetics techniques (Feng et al., 2015, J Virol, 89: 6860-6873) andgrown in MDCK cells at 33° C. Influenza A/Ca/IL/41915/2015 H3N2,recently isolated from the US 2015 outbreak, was also grown in MDCKcells at 33° C. For infections, virus stocks were diluted in phosphatebuffered saline (PBS), 0.3% bovine albumin (BA) and 1% PS (PBS/BA/PS).After viral infections, cells were maintained in DMEM with 0.3% BA, 1%PSG, and 1 μg/ml TPCK-treated trypsin (Sigma) (Zhou et al., 2012,Vaccine, 30: 3691-3702).

Plasmids

To generate the recombinant H3N8 LACIV, the PB2 and PB1 genes weresubcloned in a pUC19 plasmid (New England Biolabs) and then, ismutations (PB2 N265S; and PB1 K391E, D581G, and A661T) present in thehuman A/AA/6/60 H2N2 LAIV were introduced by site-directed mutagenesisusing specific primers. The presence of introduced mutations wasconfirmed by sequencing. Mutated PB2 and PB1 viral segments weresubcloned from the pUC19 into the ambisense pDZ plasmid for virusrescue. To test the ability of WT and LACIV H3N8 polymerases toreplicate and transcribe at different (33° C., 37° C. and 39° C.)temperatures using a minigenome assay, we engineered a pPolI plasmidcontaining the canine RNA polymerase I (Pol-I) promoter and the mousePol-I terminator separated by SapI endonuclease restriction sites(cpPol-I). The canine Pol-I promoter was obtained by PCR from MDCK cells(Murakami et al., 2008, J Virol, 82: 1605-1609). Then, the Gaussialuciferase (Gluc) reporter gene containing the 3′ and the 5′ non-codingregions of the viral NP (v) RNA was cloned into the cpPol-I plasmid togenerate the cpPol-I Gluc reporter plasmid. All plasmids were confirmedby sequencing (ACGT, Inc).

The nucleotide sequences for each segment, and amino acid sequences foreach encoded protein, of the H3N8 CIV, are provided in SEQ ID NOs: 5-22.The mutated sequences for segment 1, PB2 protein, segment 2, and PB1protein, are provided in SEQ ID NOs: 1-4, FIG. 13, and FIG. 14.

Minigenome Assays

For the minigenome assays, parental MDCK cells (12-well plate format,5×10⁵ cells/well, triplicates) were co-transfected in suspension usingLipofectamine 2000 with 250 ng of each of the H3N8 WT or LACIV ambisensepDZ PB2, PB1, PA and NP plasmids, together with 500 ng of the cpPol-IGluc plasmid. A mammalian expression pCAGGS plasmid encoding Cypridinaluciferase (Cluc, 100 ng) was also included to normalize transfectionefficiencies (Cheng et al., 2015, J Virol, 89: 3523-3533). Cellstransfected in the absence of the pDZ NP plasmid were used as negativecontrol. At 24 hours post-transfection, Gluc and Cluc expression levelswere determined using Luciferase Assay kits (New England BioLabs) andquantified with a Lumicount luminometer (Packard). Fold induction overthe level of induction for the negative control (absence of NP) wasdetermined. The mean values and standard deviations (SDs) werecalculated and statistical analysis was performed using a two-tailedStudent t test using Microsoft Excel software.

Virus Rescue

Virus rescues were performed as previously described (Nogales et al.,2014, Virology, 476C: 206-216, Nogales et al., 2014, J Viro, 88:10525-10540). Briefly, co-cultures (1:1) of 293T/MDCK cells (6-wellplate format, 10⁶ cells/well, triplicates) were co-transfected insuspension, using Lipofectamine 2000 (Invitrogen), with 1 μg of theeight-ambisense H3N8 WT CIV (pDZ-PB2, -PB1, -PA, -HA, -NP, -NA, -M and-NS) plasmids. To rescue the H3N8 LACIV, WT PB2 and PB1 pDZ plasmidswere substituted by those containing PB2 and PB1 H3N8 LACIV. At 12 hourspost-transfection, transfection medium was replaced with post-infection(p.i.) medium containing DMEM supplemented with 0.3% BSA, 1% PSG, and0.5 μg/ml TPCK-treated trypsin (Sigma). Tissue culture supernatants(TCS) were collected at 3 days post-transfection, clarified, and used toinfect fresh monolayers of MDCK cells (6-well plate format, 10⁶cells/well, triplicates). At 3 days p.i., recombinant viruses wereplaque purified and scaled up using MDCK cells at 33° C. (Nogales etal., 2014, J Viro, 88: 10525-10540). Virus stocks were titrated bystandard plaque assay (plaque forming units, PFU/ml) in MDCK cells at33° C. (Nogales et al., 2014, J Viro, 88: 10525-10540).

Virus Growth Kinetics

Multicycle growth analyses were performed by infecting confluentmonolayers of MDCK cells (12-well plate format, 5×10⁵ cells/well,triplicates) at a multiplicity of infection (MOI) of 0.001. Viral titersin TCS collected at various times p.i. were determined by immunofocusassay (fluorescent forming units, FFU/ml) in MDCK cells as previouslydescribed (Nogales et al., 2014, J Viro, 88: 10525-10540). Briefly,confluent MDCK cells (96-well plate format, 5×10⁴ cells/well,triplicates) were infected with 10-fold serial dilutions of H3N8 WT orLACIV. At 12 hours p.i., cells were fixed and permeabilized (4%formaldehyde, 0.5% Triton X-100 in PBS) for 15 minutes at roomtemperature. After washing with PBS, cells were incubated in blockingsolution (2.5% BSA in PBS) for 1 hour at room temperature and thenincubated with 1 μg/ml of an anti-NP monoclonal antibody (HB-65, ATTC)for 1 hour at 37° C. After washing with PBS, cells were incubated withFITC-conjugated secondary anti-mouse antibody (Dako) for 1 hour at 37°C. The mean values and standard deviation (SDs) were calculated usingMicrosoft Excel software.

Mice Experiments

Adult (5- to 7-week-old) female WT C57BL/6 mice were purchased from theNational Cancer Institute (NCI) and maintained under specificpathogen-free conditions. Mice were anesthetized intraperitoneally(i.p.) with 2,2,2-tribromoethanol (Avertin; 240 mg/kg of body weight)and then inoculated intranasally (i.n.) with 30 μl of the indicatedamounts of H3N8 WT or LACIV. Alternatively, 100 μl of a commerciallyavailable inactivated H3N8 CIV vaccine (Nobivac, Merck Animal Health) orinactivated H3N2 CIV vaccine (Novartis) were inoculated intramuscularly(i.m). Virus replication was determined by measuring viral titers in thelungs of infected mice at the indicated days p.i. To that end, threemice in each group were euthanized by administration of a lethal dose ofavertin and exsanguination, and lungs were collected and homogenized.Virus titers were determined by immunofocus assay (FFU/ml) as indicatedabove. Mice sera were collected by submandibular bleeding 24 hours priorto viral challenges and evaluated for the presence of influenza virusantibodies by enzyme-linked immunosorbent assays (ELISA) andneutralizing antibodies by hemagglutination inhibition (HAI) assays.

ELISAs

ELISAs were performed as previously described (Nogales et al., 2014, JViro, 88: 10525-10540) by coating 96-well plates at 4° C. for 16 hourswith lysates from mock, H3N8 or H3N2 WT CIV-infected MDCK cells. Afterblocking with 1% BSA for 1 hour at room temperature, plates wereincubated with 2-fold serial dilutions (starting dilution of 1:50) ofmice sera for 1 hour at 37° C. After incubation, plates were washed withH₂O, and incubated with a HRP-conjugated goat anti-mouse IgG (1:2,000;Southern Biotech) for 1 hour at 37° C. Reactions were then developedwith tetramethylbenzidine (TMB) substrate (BioLegend) for 10 minutes atroom temperature, quenched with 2N H2504, and read at 450 nm (Vmaxkinetic microplate reader; Molecular Devices).

HAI Assays

To evaluate the presence of H3N8 CIV neutralizing antibodies, mice serawere treated with receptor-destroying enzyme (RDE; Denka Seiken) andheat inactivated for 30 min at 56° C. Sera were then serially 2-folddiluted (starting dilution of 1:50) in 96-well V-bottom plates and mixed1:1 with 4 hemagglutinating units (HAU) of WT H3N8 CIV for 30 min atroom temperature. The HAI titers were determined by adding 0.5% turkeyred blood cells (RBCs) to the virus-antibody mixtures for 30 min on ice,as previously described (Nogales et al., 2014, J Viro, 88: 10525-10540).The GMT and SDs from individual mice were calculated from the last wellwhere hemagglutination was inhibited.

Canine Tracheal Explants Preparation

Three dog tracheas were collected from healthy Beagles (Charles RiverLaboratories) used as negative controls in other unrelated researchstudies. Briefly, tracheas were collected aseptically immediately uponeuthanasia and transported in pre-warmed medium as described previously(Gonzalez et al., 2014, J Virol, 88(16): 9208-19). Tracheas were washeda minimum of 5 times for a total period of 4 hours and maintained at 33°C., 5% CO₂, and 95% humidity between washes. The connective tissue wasremoved and the trachea was then open lengthwise. Each tracheal ring wasdivided in four 0.25 cm² explants and transferred epithelium facingupwards onto an agarose plug covered by a sterile filter. The explantswere then kept for a total of 6 days at 33° C., 5% CO₂, and 95%humidity.

Infection of Explants and Quantification of Virus Titers

Explants were infected after a period of 24 hours post-dissection(designed as day zero) with 200 PFU of WT H3N8 CIV, H3N8 LACIV or mockinfected with culture medium. Inoculated explants were sampled for virusquantification, bead clearance assays, and histology at day 0, 1, 3 and5 post-infection. Viral replication was evaluated by plaque assays onMDCK cells by means of immunostaining of plaques.

Estimation of Bead Clearance Time

The ciliary function of tracheal explants was evaluated as describedpreviously (Gonzalez et al., 2014, J Virol, 88(16): 9208-19) by placingfive microliters of polystyrene microsphere beads (Polysciences,Northampton, UK) on the explant apical surface and measuring the time todisplace the beads.

Histological Analysis and Immunohistochemistry

After collection, the explants were fixed in 10% buffered formalin for aminimum of 48 hours, before paraffin embedding. Subsequently, 4 μmsections of paraffin embedded tissue were either stained withHaematoxylin and Eosin for histopathological evaluation or immunostainedfor the viral nucleoprotein (NP) using standard procedures. For NPimmunostaining, the Dako supervision system was used following themanufacturer's protocol, along with a monoclonal mouse anti-NP (cloneHB65; dilution 1:500). Slides were counterstained with Mayer'shaematoxylin. Histological images were captured with the cellD software(Olympus).

The results of the experiments are now described

Generation and Characterization of the Ts H3N8 LACIV

To generate a ts H3N8 LACIV the five mutations in the PB2, PB1, and NPgenes that were previously identified as the major determinantsresponsible for the ts phenotype of the human A/AA/6/60 H2N2 LAIV wereintroduced (Cox et al., 1988, Virology, 167: 554-567, Jin et al., 2003,Virology, 306: 18-24). These amino acid mutations include one mutationin PB2 (N2655) and three mutations in PB1 (K391E, D581G, A661T) (FIG.1A). No mutation was introduced into the viral NP since H3N8 CIV alreadycontains a G at position 34.

To determine if the mutations introduced into the PB2 and PB1 genesconfer a ts phenotype to the H3N8 CIV polymerase, a minigenome assay wasperformed. To that end, a pPol plasmid containing a vRNA-like segmentencoding Gluc under the control of a canine (c) PolI promoter wasco-transfected in MDCK cells with the ambisense pDZ plasmids expressingthe viral PB2 (WT or LACIV), PB1 (WT or LACIV), PA and NP. Aftertransfection, cells were incubated at different temperatures (33° C.,37° C. and 39° C.) and at 24 hours post-transfection, Gluc expression inthe TCS was quantified. Both, WT and LACIV H3N8 resulted in similar Glucexpression levels at 33° C. (FIG. 1B). However, a reduction of Glucexpression was observed at higher temperatures (37° C. and 39° C.) incells transfected with the H3N8 LACIV plasmids, indicating that themutations responsible for the ts phenotype of A/AA/6/60 H2N2 alsoresulted in a ts phenotype when introduced in the H3N8 CIV, aspreviously described for other viruses.

Next an H3N8 LACIV was generated using plasmid-based reverse geneticapproaches, as previously described. To test if the ts mutationsintroduced in the H3N8 CIV polymerases can also result in impairedgrowth of the H3N8 LACIV at restrictive (37° C. and 39° C.) temperaturesbut not permissive (33° C.) temperatures, the replication kinetics ofthe H3N8 LACIV was evaluated and compared to that of the WT H3N8 CIV inMDCK cells infected at low (0.001) MOI (FIG. 2). At 33° C., both WT andLACIV H3N8 grew with undistinguishable kinetics and reached similar hightiters (10⁷ FFU/ml) at 48-72 h p.i. (FIG. 2A). However, at higher (37°C. and 39° C.) temperatures, the WT H3N8 CIV replicated at similarlevels as those observed at 33° C. while replication of the H3N8 LACIVwas impaired ˜2-3-logs at 37° C. (FIG. 2B) or was not detected at 39° C.(FIG. 2C). Altogether, these data demonstrate that the PB2 and PB1mutations responsible of the ts phenotype of the A/AA/6/60 H2N2 humanLAIV are able to confer also a ts phenotype to the H3N8 CIV and that arecombinant H3N8 CIV containing these mutations has a ts phenotype butable to propagate at levels compared to those of WT H3N8 at 33° C.,which is important for vaccine production, as in the case of the humanLAIV.

LACIV H3N8 is Attenuated In Vivo

As the H3N8 LACIV presented defects in replication at higher (37° C. and39° C.) temperatures, it was examined whether the virus was alsoattenuated in mice. No signs or symptoms of infection were detectedafter the infection with WT H3N8 CIV. Therefore, CIV replication wasmeasured as an attenuation index. To this end, groups of mice (N=6) wereinoculated i.n. with 10⁵ PFU of WT or LACIV H3N8 and viral titers in thelungs of infected mice were evaluated on days 2 (N=3) and 4 (N=3) p.i.(FIG. 3). Notably, virus replication in the lungs was only detected inmice infected with WT H3N8 CIV and no virus was detected in miceinfected with the H3N8 LACIV. Altogether, these results indicate thatH3N8 LACIV is also attenuated in vivo.

Vaccination with H3N8 LACIV Induces Protective Immunity Against WT H3N8CIV Challenge

Since H3N8 LACIV was attenuated in mice, as compared to the WT H3N8 CIV,it was examined whether H3N8 LACIV can be implemented as a LAIV for thetreatment of H3N8 CIV. To evaluate this possibility, mice (N=6) werevaccinated (i.n.) with 10³ PFU of H3N8 WT or LACIV. In addition, a groupof mice was mock (PBS) vaccinated or vaccinated intramuscularly (i.m.)with 100 μl of Nobivac, a commercially available vaccine against H3N8CIV. Then, humoral immune responses were evaluated in sera collected 2weeks after vaccination (FIG. 4A). Total H3N8 CIV antibody responseswere characterized by ELISA using cell lysates from mock- or H3N8CIV-infected MDCK cells (Nogales et al., 2016, J Virol, 90: 6291-6302).Mice vaccinated with the H3N8 LACIV elicited high serum IgG titersagainst parental H3N8 CIV. However, antibody titers of mice vaccinatedwith the IIV Nobivac were significantly reduced as compared with thosefrom H3N8 LACIV or WT vaccinated mice (FIG. 4A), indicating that H3N8LACIV induces strongest humoral responses than the IIV, similar to thesituation previously described with other influenza viruses.Additionally, HAI assays were performed to examine the presence ofneutralizing antibodies on sera from vaccinated mice (Table 3). Asexpected protective antibody titers against CIV H3N8 were higher in micevaccinated with the H3N8 LACIV than those observed with the H3N8 IIVNobivac.

Next, experiments were conducted to evaluate the ability of H3N8 LACIVto induce protective immunity. To that end, mice (N=6) were vaccinatedi.n. with 10³ PFU of H3N8 WT or LACIV, i.m. with 100 μl of the IIVNobivac, or mock (PBS) vaccinated. Two weeks post-vaccination, mice werechallenged with 10⁵ PFU of homologous WT H3N8 CIV and viral titers inthe lungs of infected mice (N=3/group) were evaluate at days 2 and 4post-challenge (FIG. 4B). As expected, mock-vaccinated mice showed highviral titers at days 2 and 4 p.i. Importantly, lungs from mice immunizedwith H3N8 WT CIV and with LACIV showed no detectable virus titers ateither day post-challenge (FIG. 4B). However, mice vaccinated with theH3N8 IIV Nobivac showed high viral titers at day 2, although nodetectable virus at day 4 post-infection (FIG. 4B). Altogether, thesedata indicated that H3N8 LACIV vaccination induce better immuneresponses, including neutralizing antibodies, than mice vaccinated withthe H3N8 IIV Nobivac, resulting in better protection efficacy against WTH3N8 CIV challenge, favoring the implementation of the H3N8 LACIV overthe IIV for a better protection against H3N8 CIV.

H3N8 LACIV is Attenuated in Canine Tracheal Explants Compared to H3N8 WTCIV

To compare H3N8 LACIV and H3N8 WT CIV pathogenicity and replicationefficiency at the site of infection within the natural host, dogtracheal explants were infected with each virus and histological lesions(FIG. 5A), changes in ciliary function (FIG. 5C), viral replication(FIG. 5B) and viral Nucleoprotein (NP) expression (FIG. 5D) werecompared at different times post-infection. H3N8 WT CIV induced majorhistological changes in dog tracheal explants, with thinning anddesquamation of the epithelium, loss of cilia (FIG. 5A), and significantreduction of ciliary function (FIG. 5C) from day 1 to day 5 postinfection. Interestingly, histological damages induced by H3N8 LACIVwere delayed and reduced compared to WT CIV, as the epitheliummaintained its normal thickness until day 3 post infection (FIG. 5A) andthe ciliary function (FIG. 5C) was only significantly reduced from day 3post infection.

Additionally, viral kinetics and NP expression were comparable betweenthe two viruses, although only CIV WT was detectable at day 1 postinfection (FIG. 5D). Overall, these results indicate that LACIVpathogenicity is attenuated in canine tracheal explants compared to H3N8CIV WT.H3N8 LACIV Provides Limited Protection Against Heterologous H3N2 CIV

Next, it was evaluated if H3N8 LACIV can induce protective immunityagainst a heterologous H3N2 CIV challenge (FIG. 6). To that end, mice(N=6) were vaccinated (i.n.) with 10³ PFU of H3N8 CIV WT or LACIV. Asinternal controls, a group of mice was mock (PBS) vaccinated orvaccinated (i.m.) with 100 μl of the H3N8 IIV Nobivac or a commercialH3N2 IIV (Zoetis). Then, presence of antibodies against H3N2 CIV wasevaluated by ELISA using cell lysates from mock- or H3N2 CIV-infectedMDCK cells (FIG. 6A). Antibodies against H3N2 CIV were detected in serafrom mice vaccinated with WT H3N8 CIV and, to a lower extent, in micevaccinated with H3N8 LACIV, although the levels were lower than thoseobtained against H3N8 CIV (FIG. 4). No detectable IgG antibodies againstH3N2 CIV were detected in mice vaccinated with the H3N8 IIV Nobivac. Asexpected, the H3N2 CIV IIV induced higher IgG antibodies against H3N2CIV. These lower level of cross-reactive antibodies against H3N2 CIVupon vaccination with the H3N8 LACIV were further confirmed afterchallenge (i.n.) with 10⁵ PFU H3N2 CIV 2 weeks post-vaccination (FIG.6B). Mock-vaccinated mice showed high H3N2 CIV titers that wereundistinguishable, either at 2 or 4 days post-challenge, from theanimals vaccinated with the H3N8 CIV IIV Nobivac. On the other hand,mice vaccinated with the H3N2 CIV IIV showed reduced or undetectabletiters, respectively, at day 2 and 4 post-challenge. Notably, althoughwe observed similar H3N2 CIV titers at day 2 post-challenge, viraltiters at day 4 post-infection in mice vaccinated with the H3N8 LACIVwere ˜100 times lower than those obtained in the mock vaccinated group.These results indicate that although H3N8 LACIV can induce somecross-reactive immune responses and protection efficacy against H3N2CIV, the efficacy of the H3N8 LACIV is lower than that obtained with theH3N2 IIV.

A Novel LAIV for the Treatment of H3N8 CIV

In this work, we have developed, for the first time, a novel LAIV forthe treatment of H3N8 CIV. Using plasmid-based reverse geneticstechniques, we have generated a recombinant H3N8 CIV containing themutations responsible for the ts, ca, att phenotype of the humanA/AA/6/60 H2N2 LAIV. Introduction of these mutations in the H3N8 CIVresulted in a ts H3N8 CIV that was highly attenuated, as compared toH3N8 CIV WT, in replication in vivo but able to confer, upon a singlei.n. immunization, complete protection against challenge with WT H3N8CIV, demonstrating the feasibility of implementing the ts H3N8 LACIV asa safe, immunogenic and protective LAIV for the treatment of H3N8 CIVinfections.

The ts, ca, att A/AA/6/60 H2N2 LAIV has been licensed for human use.This A/AA/6/60 H2N2 LAIV is used as a master donor virus (MDV) for thegeneration of both seasonal or potentially pandemic human LAIV bycreating reassortant viruses containing the six internal vRNA segments(PB2, PB1, PA, NP, M, and NS) from A/AA/6/60 H2N2 LAIV, responsible forthe attenuated phenotype, and the two glycoprotein encoding vRNAs (HAand NA) from a virus that antigenically matches the strains predicted tocirculate in the upcoming influenza season (in the case of a seasonalvaccine) or potentially pandemic strains (in the case of the pandemicvaccine) (Maassab, 1999, Reviews in medical virology, 9: 237-244, Murphyet a., 2002, Viral immunology, 15: 295-323.). It has been previouslyshown that five ts mutations (PB2 N265S; PB1 K391E, D581G, A661T; and NPD34G) are responsible for the ts, ca, att phenotype of the A/AA/6/60H2N2 LAIV. Moreover, introduction of these mutations in other influenzaviruses has been shown to be sufficient to impart a strong ts phenotypeand attenuation in other viral strains, such as PR8 (Cox et al., 2015, JVirol, 89(6): 3421-3426, Jin et al., 2004, J Virol, 78: 995-998) andpH1N1 (Zhou et al., 2012, Vaccine, 30: 3691-3702).

Intranasal immunization is a desirable delivery method to preventinfection with IAV because it leads to the generation of a mucosalimmune responses, creating an immune barrier at the site of potentialinfection (Kohlmeier et al., 2009, Annual review of immunology, 27:61-82). Indeed, LAIVs elicit not only a robust systemic humoral responsebut also a mucosal immune response (Cheng et al., 2013, The Journal ofinfectious diseases, 208: 594-602, De Villiers et al., 2009, Vaccine,28: 228-234, Katsura et al., 2012, Vaccine, 30: 6027-6033, Murphy etal., 2002, Viral immunology, 15: 295-323, Victor et al., 2012, J Virol,86(8): 4123-4128). Similar to infection with WT IAV, it has been showedthat LAIV immunization also leads to recruitment of influenza-specificCD8 T cells to the lungs (Baker et al., 2013, J Virol, 87: 8591-8605,Guo et al., 2014, J Virol, 88: 12006-12016, Katsura et al., 2012,Vaccine, 30: 6027-6033, Powell et al., 2012, J Virol, 86: 13397-13406,Uraki et al., 2013, J Virol, 87: 7874-7881), which is likely to be themain contributor of immunity against heterologous influenza challenge(Baker et al., 2013, J Virol, 87: 8591-8605, Guo et al., 2014, J Virol,88: 12006-12016). Thus, a LAIV rather than IIV is desired for thecontrol of IAV infections.

Since the emergence of H3N8 CIV in 1999 in the USA and the H3N2 CIV inAsia in 2005, CIVs have been circulating in the dog populations,particularly in shelters (Crawford et al., 2005, Science, 310: 482-485,Holt et al., 2010, Journal of the American Veterinary MedicalAssociation, 237: 71-73). Indeed, H3N8 and H3N2 CIVs are routinelyisolated from such facilities (Hayward et al., 2010, J Virol,84:12636-12645, Pecoraro et al., 2013, Journal of veterinary diagnosticinvestigation, 25: 402-406, Rivailler et al., 2010, Virology, 408:71-79). Notably, H3N2 CIV has appeared to not be limited to Asia, andrecently (2015) has been imported to the USA. Importantly, H3N8 and H3N2CIVs not only represent a new threat to canine health, since they couldovercome the species barrier and infect humans or other species. Infact, the H3 subtype of IAV are able to infect multiples species,including humans, pigs, horses, dogs, cats, seals, poultry and wildaquatic birds (Bean et al., 1992, J Virol, 66:1129-1138, Both et al.,1983, J Virol, 48:52, Bush et al., 1999, Molecular biology andevolution, 16: 1457-1465, de Jong et al., 2007, J Virol, 81: 4315-4322,Epperson et al., 2013, Clinical infectious diseases, 57 Suppl 1:S4-S11,Rivailler et al., 2010, Virology, 408: 71-79, Song et al., 2008,Emerging infectious diseases, 14: 741-746). Moreover, it has been shownthe possibility of H3N8 CIV to reassort with H1N1 IAV (Gonzalez et al.,2014, J Virol, 88: 9208-9219). Furthermore, reassortment between CIVsand human IAVs is not without precedent, as a naturally occurring H3N1virus carrying the HA gene of an avian-like H3N2 CIV and the other sevensegments of the human pH1N1 has been reported (Song et al., 2012, TheJournal of general virology, 93: 551-554). Therefore, dogs could act asan intermediate host for genetic reassortment between mammalian(including human) and avian IAVs, facilitating the generation of newIAVs with pandemic potential for humans. To date, no transmission ofH3N8 or H3N2 CIV transmission from dogs to humans have been reported.

There is an opportunity to control or even eradicate H3N8 and H3N2 CIVsfrom the dog population throughout vaccination, therefore reducing thepossibility of their transmission into humans or the risk of generating,by reassortment with other IAVs, new viral strains with a pandemicpotential for humans. Currently, only IIV are available for thetreatment of H3N8 or H3N2 CIV infections but their efficacy is limited.Thus, the generation and implementation of CIV LAIVs represent a betteroption for the treatment of CIV infections since they afford better andfaster induction of adaptive immune responses, as it has been shown withhuman influenza vaccines (Belshe et al., 2007, The New England journalof medicine, 356: 685-696). Moreover, they also represent an excellentoption for the potential eradication of CIVs from the dog population,before they jump to other animal species. Successful CIV LAIV candidatesmust show in vivo attenuation, while retaining immunogenicity andprotection efficacy, and must also grow well in manufacturing-suitabletissue culture platforms (Hussain et al., 2010, Vaccine, 28: 3848-3855,Pica et al., 2013, Annual review of medicine, 64: 189-202).

In this study the feasibility of generating an H3N8 LACIV isdemonstrated by introducing the four amino acid changes present in theviral polymerase PB2 and PB1 into the backbone of the H3N8 CIV. It isshown that the H3N8 LACIV replicates efficiently in vitro at permissivelow (33° C.) temperatures but is restricted at high (37° C. and 39° C.)temperatures (FIG. 1 and FIG. 2). Importantly, the H3N8 LACIV wasattenuated, as compared with H3N8 CIV WT, in the lungs of infected mice(FIG. 3) but able to induce protective immune responses againstchallenge with homologous H3N8 CIV challenge (FIG. 4). Remarkably, H3N8LACIV elicited better humoral responses and protection than thatobtained with a commercial H3N8 IIV (Nobivac). However, the H3N8 LACIVinduced low levels of cross-reactive antibodies and limited protectionefficacy against an heterologous challenge with H3N2 CIV (FIG. 6),demonstrating the need of generating an H3N2 LACIV for the treatment ofH3N2 CIV infections in dogs.

TABLE 3 Immunogenicity of LACIV and sciCIV Immunization and dose^(a)Mean (SD) serum HAI titer^(b) PBS — ≤8 (ND) WT  10³ PFU 215.3 (64) LACIV  10³ 76.1 (32) Nobivac 100 μl 26.9 (8)  ^(a)Virus was administeredintranasally to anesthetized mice (n = 4), Nobivac was administeredintramuscularly, and sera were collected at 14 days postinfection.^(b)Four HAU of the WT virus was incubated with 2-fold serial dilutionsof the indicated sera. ND, not determined.

Example 2: Development and Characterization of a Live AttenuatedInfluenza Vaccine (LAIV) Against H3N2 CIV Based on a TemperatureSensitive (Ts) Mutant

Described herein are experiments used to develop, generate, andcharacterize, a LAIV for CIV H3N2. The H3N2 LAIV presented herein isbased on the CIV H3N8 LAIV, described in Example 1, which was used as amaster donor virus (MDV) to express the HA and NA of CIV H3N2. As, thetemperature sensitive H3N8 LAIV has demonstrated to be safe, it can beused as an MDV to express viral proteins from other circulating strainsto update the LAIV to protect against new strains.

The nucleotide sequences for each segment, and amino acid sequences foreach encoded protein, of the H3N8 CIV, are provided in SEQ ID NOs: 5-22.The mutated sequences for segment 1, PB2 protein, segment 2, and PB1protein, are provided in SEQ ID NOs: 1-4, FIG. 13, and FIG. 14. Thenucleotide sequences for segment 4 and segment 6, and the amino acidsequences of the encoded HA and NA, of the H3N2 CIV, used in thedevelopment of the H3N2 LAIV described herein are provided in SEQ IDNOs: 23-26.

FIG. 7 is a schematic depicting the generation of the CIV H3N2 LAIV,based upon the CIV H3N8 LAIV described in Example 1. As described above,the CIV H3N8 LAIV comprises specific amino acid substitutions in PB1 andPB2. Amino acid substitutions N265S (PB2) and K391E, E581G, and A661T(PB1) were introduced into the A/canine/NY/dog23/2009 H3N8 (CIV H3N8) togenerate the CIV H3N8 LAIV. CIV H3N8 LAIV was used as a master donorvirus (MDV) to generate the CIV H3N2 LAIV that contains the internalviral segments (PB2, PB1, PA, NP, M and NS) of CIV H3N8 LAIV and the HAand NA of A/Ca/IL/41915/2015 H3N2 (CIV H3N2).

Experiments were then conducted to examine the growth kinetics of theCIV H3N2 LAIV. Canine MDCK cells (12-well plate format, 5×10⁵cells/well, triplicates) were infected at low multiplicity of infection(MOI, 0.001) with A/Canine/Illinois/11613/2015 H3N2 (CIV H3N2 WT),A/Canine/NY/Dog23/2009 H3N8 (CIV H3N8 WT) and the two LAIVs (CIV H3N2LAIV and CIV H3N8 LAIV) and incubated at 33° C. (FIG. 8A), 37° C. (FIG.8B) and 39° C. (FIG. 8C). Tissue culture supernatants were collected at12, 24, 48, 72 and 96 hours post-infection. Viral titers in tissueculture supernatants were determined by immunofocus assay (Focus FormingUnits, FFU/ml) using an anti-NP monoclonal antibody (HT-103). It wasobserved that the H3N2 LAIV displayed similar growth, as compared to WT,at 33° C., but is attenuated at 37° C. and 39° C. (FIG. 8A-FIG. 8C).

Next, experiments were conducted to examine the attenuation of CIV H3N2LAIV. Female 6-to-8-week-old C57BL/6 WT mice (N=6) were infectedintranasally (i.n.) with 1×10⁵ FFU of CIV H3N2 WT or CIV H3N2 LAIV.Presence of viruses in the lungs (FIG. 9A) and the nasal mucosal (FIG.9B) of infected mice were evaluated at days 2 (N=3) and 4 (N=3)post-infection by immunofocus assay (FFU/ml) using an anti-NP monoclonalantibody (HT-103). Significantly less virus was detected in the lungs ofmice infected with the H3N2 LAIV, as compared to H3N2 WT (FIG. 9A).

Next, experiments were conducted examining the induction of humoralresponses by CIV H3N2 LAIV. Female 6-to-8-week-old C57BL/6 WT mice (N=6)were immunized with 1×10³ FFU of CIV H3N2 WT or CIV H3N2 LAIV. Mice werealso mock vaccinated or vaccinated with 100 μl/mice of an inactivatedCIV H3N2 vaccine (Zoetis) as negative and positive controls,respectively. At 14 days post-vaccination, mice were bled and the serawere collected and evaluated individually by ELISA for IgG antibodiesagainst total influenza virus protein using cell extracts of MDCK cellsinfected with A/Canine/Illinois/11613/2015 H3N2 WT virus (FIG. 10A) orA/Canine/NY/Dog23/2009 H3N8 WT virus (FIG. 10B). Mock-infected cellextracts were used to evaluate the specificity of the antibody response.It is demonstrated that the H3N2 LAIV induced a greater H3N2 specificimmune response, as compared to the inactivated H3N2 CIV, and comparableto H3N2 WT (FIG. 10A).

Next, experiments were conducted examining the protection efficacy ofCIV H3N2 LAIV. Female 6-to-8-week-old C57BL/6 WT mice (N=12) werevaccinated with 1×10³ FFU of CIV H3N2 WT or CIV H3N2 LAIV. Mice werealso mock vaccinated or vaccinated with 100 ul/mice of a CIV H3N2inactivated vaccine (Zoetis) as negative and positive controls,respectively. Two weeks post-vaccination, mice (N=6) were challengedwith 1×10⁵ FFU of CIV H3N2 WT (FIG. 11A) or CIV H3N8 WT (FIG. 11). Viraltiters of challenged viruses at days 2 (N=3) and 4 (N=3) post-infectionwere evaluated from lung homogenates by immunofocus assay (FFU/ml) usingan anti-NP monoclonal (HT-103 or HB-65 respectively). It is observedthat the vaccination with H3N2 LAIV completely protected against H3N2challenge, and was improved over results from mice vaccinated withinactivated H3N2 CIV (FIG. 11A).

Based on the promising results with the CIV H3N8 LAIV and CIV H3N2LAIVs, it is examined whether vaccination with both CIV H3N8 and H3N2LAIVs confer protection against challenge against both CIVs for itsimplementation as a bivalent vaccine (FIG. 12)

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variations.

What is claimed is:
 1. An immunological composition comprising alive-attenuated canine influenza virus (LACIV), wherein the LACIVcomprises segment 1 of the viral genome encoding mutant PB2 comprisingthe amino acid sequence of SEQ ID NO: 3; and segment 2 of the viralgenome encoding mutant PB1 comprising the amino acid sequence of SEQ IDNO:
 4. 2. The composition of claim 1, wherein the LACIV temperaturesensitive such that the LACIV exhibits reduced viral replication ascompared to wildtype canine influenza virus at a temperature selectedfrom the group consisting of normal body temperature and elevated bodytemperature.
 3. The composition of claim 1, wherein the segment 1comprises the nucleic acid sequence set forth in SEQ ID NO:
 1. 4. Thecomposition of claim 1, wherein the segment 2 comprises the nucleic acidsequence set forth in SEQ ID NO:
 2. 5. The composition of claim 1wherein the LACIV is derived from H3N8 subtype of influenza A virus. 6.The composition of claim 1, wherein the LACIV expresses HA and NA ofH3N8.
 7. The composition of claim 1, wherein the LACIV expresses HA andNA of H3N2.
 8. The composition of claim 1 wherein the composition isused for the treatment or prevention of canine influenza in a subject.9. A method for treating or preventing canine influenza in a subject,the method comprising administering to the subject the immunologicalcomposition of claim
 1. 10. The method of claim 9, wherein the subjectdoes not have canine influenza, and wherein the method induces immunityagainst one or more of: influenza A virus subtype H3N8 and influenza Avirus subtype H3N2.
 11. The method of claim 9, wherein the subject isinfected with at least one or more of: influenza A virus subtype H3N8and influenza A virus subtype H3N2; and wherein the method induces atherapeutic immune response.
 12. The method of claim 9, wherein theimmunological composition is administered intranasally, intratracheally,orally, intradermally, intramuscularly, intraperitoneally,intravenously, or subcutaneously.
 13. The method of claim 9, wherein thesubject is a dog.